Polypeptides, their production and use

ABSTRACT

The present invention relates to the ligand polypeptide for the human pituitary- and mouse pancreas-derived G protein-coupled receptor proteins. The ligand polypeptide or the DNA which codes for the ligand polypeptide can be used for (1) development of medicines such as pituitary function modulators, central nervious system function modulators, and pancreatic function modulators, and (2) development of receptor binding assay systems using the expression of recombinant receptor proteins and screening of pharmaceutical candidate compounds. In particular, by the receptor binding assay systems utilizing the expression of recombinant G protein-coupled receptor proteins in accordance with the invention, agonists and antagonists of G protein-coupled receptors which are specific to human and other warm-blooded animals can be screened and the agonists or antagonists obtained can be used as therapeutic and prophylactic agents for various diseases.

TECHNICAL FIELD

The present invention relates to a novel ligand polypeptide for the Gprotein-coupled receptor protein and a DNA comprising a DNA encoding theligand polypeptide.

BACKGROUND ART

Many hormones and neurotransmitters mediate biological functions throughspecific receptors present on the cell membrane. Many of these receptorsengage themselves in the intracellular transduction of signals throughactivation of the coupled guanine nucleotide-binding protein(hereinafter sometimes referred to briefly as G protein) and have thecommon structure comprising 7 transmembrane domains. Therefore, thesereceptors are collectively referred to as G protein-coupled receptor or7-transmembrane receptor.

One of the pathways to modulate biological functions mediated by suchhormones or neurotransmitters through G protein-coupled receptors is thehypothalamo-pituitary system. Thus, the secretion of pituitary hormonefrom the hypophysis is controlled by hypothalamic hormones(pituitatropic releasing factor) and the functions of the target cellsor organs are regulated through the pituitary hormones released into thecirculation. This pathway carries out functional modulations ofimportance to the living body, such as homeostasis and regulation of thereproduction, development, metabolism and growth of individuals. Thesecretion of pituitary hormones is controlled by a positive feedback ora negative feedback mechanism involving hypothalamic hormone and theperipheral hormone secreted from the target endocrine gland. The variousreceptor proteins present in the hypophysis are playing a central rolein the regulation of the hypothalamus-pituitary system.

Meanwhile, it is known that these hormones and factors as well as theirreceptors are not localized in the hypothalamus-pituitary system but arebroadly distributed in the brain. Therefore, it is suspected that, inthe central nervous system, this substance called hypothalamus hormoneis functioning as a neurotransmitter or a neuromodulator. Moreover, thesubstance is distributed in peripheral tissues as well and thought to beplaying important roles in the respective tissue.

The pancreas is playing a crucial role in the carbohydrate metabolism bysecreting glucagon and insulin as well as digestive juice. While insulinis secreted from the pancreatic β cells, its secretion is mainlystimulated by glucose. However, it is known that β cells have a varietyof receptors and the secretion of insulin is controlled by a number offactors in addition to glucose as well as peptide hormones, e.g.galanine, somatostatin, gastric inhibitory polypeptide, glucagon,amyrin, etc.; sugars, e.g. mannose etc.; amino acids, andneurotransmitters, among others.

The means only heretofore available for identifying ligands for said Gprotein-coupled receptor proteins is estimation from the homology inprimary structure of G protein-coupled receptor proteins.

Recently, investigation for novel opioid peptides by introducing a cDNAcoding for a receptor protein which a ligand is unknown, i.e. an orphanG protein-coupled receptor protein, into animal cells have been reported(Reinsheid, R. K. et al., Science, 270, 792-794, 1995, Menular, J.-C.,et al., Nature 377, 532-535, 1995). However, in view of similarities toknown G protein-coupled receptor proteins and tissue distributions, itcould be easily anticipated in these cases that the ligand would bebelonging to the family of opioid peptides. The history of research anddevelopment in the realm of substances acting on the living body throughthe opioid receptor dates back to many years ago and various antagonistsand agonists had been developed. Therefore, among the compoundsartificially synthesized, an agonist of the receptor was picked out and,using it as a probe, expression of the receptor in the receptorcDNA-transfected cells was verified. Then, a search was made for anactivator of the intracellular signal transduction which was similar tothe agonist, the activator so found was purified, and the structure ofthe ligand was determined. However, when the homology of an orphanreceptor to known G protein-coupled receptor proteins is low, it wasvery difficult to predict its ligand.

Ligands for orphan G protein-coupled receptors expressed in thehypophysis, central nervous system, and pancreatic β cells areconsidered to be useful for developing medicines, but their structuresand functions have not been elucidated as yet.

DISCLOSURE OF INVENSION

Employing a cell in which a cDNA coding for orphan G protein-coupledreceptor protein has been expressed by a suitable means and usingmeasurement of a specific cell stimulation activity exemplified by asignal transduction activity as an indicator, the inventors of thepresent invention succeeded in screening a polypeptide which saidreceptor protein recognizes as a ligand.

Furthermore, the inventors found that a compound can be screened whichis capable of changing the binding activity of this ligand which is anactivating factor to said receptor protein.

The present invention, therefore, relates to

-   (1) A polypeptide which comprises an amino acid sequence represented    by SEQ ID NO:73 or its substantial equivalent thereto, or its amide    or ester, or a salt thereof.-   (2) The polypeptide as described in (1) above, which comprises the    amino acid sequence represented by SEQ ID NO:3, SEC ID NO:4, SEQ ID    NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID    NO:10, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ    ID NO:51, SEQ ID NO:52, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63,    SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66.-   (3) The polypeptide as described in (1) above, which comprises the    amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:44, SEQ ID    NO:45, or SEQ ID NO:59.-   (4) A partial peptide of the polypeptide as described in (1) above    its amide or ester, or a salt thereof.-   (5) A DNA which comprises a DNA having a nucleotide sequence coding    for the polypeptide as described in (1) above or the partial peptide    as described in (4) above.-   (6) The DNA as described in (5) above which comprises a nucleotide    sequence represented by SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:12, SEQ    ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,    SEQ ID NO:18, SEQ ID NO:18, SEQ ID NO:46, SEQ ID NO:53, SEQ ID    NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ    ID NO:60, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70,    SEQ ID NO:71, or SEQ ID NO:72.-   (7) A recombinant vector comprising the DNA as described in (5)    above.-   (8) A transformant carrying the DNA as described in (5) above or the    recombinant vector as described (7) above.-   (9) A method for producing the polypeptide as described in (1) above    or the partial peptide as described in (4) above, which comprises    culturing the transformant as described in (8) above.-   (10) A pharmaceutical composition containing the polypeptide, its    amide or ester as described in (1) above, or a pharmaceutically    acceptable salt thereof.-   (11) A pharmaceutical composition containing the partial peptide    peptide, its amide or ester as described in (4) above, or a    pharmaceutically acceptable salt thereof.-   (12) A pharmaceutical composition containing the DNA as described    in (5) above.-   (13) The pharmaceutical composition as described in (10), (11),    or (12) above, which is a pituitary function modulator.-   (14) The pharmaceutical composition as described in (10), (11),    or (12) above, which is a central nervous system function modulator.-   (15) The pharmaceutical composition as described in (10), (11),    or (12) above, which is a pancreatic function modulator.-   (16) An antibody against the polypeptide as described in (1) above    or against the partial peptide as described in (4) above.-   (17) A screening method for a compound capable of changing the    binding activity of the polypeptide as described in (1) above or the    partial peptide as described in (4) above, with a receptor protein    comprising an amino acid sequence represented by SEQ ID NO:21 or its    partial peptide or its substantial equivalent thereto, or a salt    thereof, which comprises making a comparison between: (i) at lease    one case where said polypeptide as described in (1) above or the    partial peptide as described in (4) above is contacted with a    receptor protein comprising an amino acid sequence represented by    SEQ ID:21 or its partial peptide or its substantial equivalent    thereto, or a salt thereof, and (ii) at least one case where said    polypeptide as described in (1) above or the partial peptide as    described in (4) above together with a sample to be tested in    contacted with protein comprising an amino acid sequence represented    by SEQ ID NO:21 or its partial peptide or its substantial equivalent    thereto, or a salt thereof.-   (18) A kit for screening for a compound capable of changing the    binding activity of the polypeptide as described in (1) above or the    partial peptide as described in (4) above with a receptor protein    comprising an amino acid sequence represented by SEQ ID NO:21 or its    partial peptide or its substantial equivalent thereto, or a salt    thereof.-   (19) A compound capable of changing the binding activity of the    polypeptide as described in (1) or the partial peptide as described    in (4) with a receptor protein comprising an amino acid sequence    represented by SEQ ID NO:21 or its partial peptide or its    substantial equivalent thereto, or a salt thereof.-   (20) A G protein-coupled receptor protein which recognizes the    polypeptide as described in (1) above or the partial peptide as    described in (4) above as a ligand, or a salt thereof.

The present invention further provides:

-   (21) the polypeptide as described in (1) above, or its amide or    ester, or a salt thereof, which comprises an amino acid sequence    selected from the group consisting of an amino acid sequence of SEQ    ID NO:73, amino acid sequences wherein 1 to 15 amino acid residues,    preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino    acid residues are deleted from the amino acid sequence of SEQ ID    NO:73, amino acid sequences wherein 1 to 80 amino acid residues,    preferably 1 to 50 amino acid residues, more preferably 1 to 10    amino acid residues are added to the amino acid sequence of SEQ ID    NO:73, and amino acid sequences wherein 1 to 15 amino acid residues,    preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino    acid resides in the amino acid sequence of SEQ ID NO:73 are    substituted with one or more other amino acid residues;-   (22) the polypeptide as described in (1) above, which comprises an    amino acid sequence wherein the peptide of SEQ ID NO:74 is added to    the N-terminus of the polypeptide comprising the amino acid sequence    of SEQ ID NO:73;-   (23) the polypeptide as described in (1) above, which in derived    from bovine, rat or human; and-   (24) the pharmaceutical composition described in (10), (11) or (12)    above, which is a therapeutic and/or prophylactic agent for    dementia, depression (melancholia), hyperkinetic    (microencephalo-pathy) syndrome, disturbance of consciousness,    anxiety syndrome, schizophrenia, horror, growth hormone secretory    disease, hyperphagia, polyphagia, hypercholesterolemia,    hyperglyceridemia, hyperlipemia, hyperprolactinemia, diabetes,    cancer, pancreatitis, renal disease, Turner's syndrome, neurosis,    rheumatoid arthritis, spinal injury, transient brain ischemia,    amyotrophic lateral sclerosis, acute myocardial infarction,    spinocerebellar degeneration, bone fracture, trauma, atopic    dermatitis, osteoporosis, asthma, epilepsy, infertility and/or    oligogalactia.

Referring to the G protein-coupled receptor protein for the ligandpolypeptide in accordance with the present invention, the inventionspecifically provides:

-   (25) the G protein-coupled receptor protein described in (20) or a    salt thereof, which comprises an amino acid sequence represented by    SEQ ID NO:19 or its substantial equivalent thereto or/and an amino    acid sequence represented by SEQ ID NO:20 or its substantial    equivalene thereto;-   (26) the G protein-coupled receptor protein described in (25) above    or a salt thereof, which comprises an amino acid sequence    represented by SEQ ID NO:21 or its substantial equivalent thereto;-   (27) the G protein-coupled receptor protein described in (25) above    or a salt thereof, which comprises an amino acid sequence    represented by SEQ ID NO:22 or its substantial equivalent thereto;-   (28) the G protein-coupled receptor protein described in (25) above    or a salt thereof, which comprises an amino acid sequence    represented by SEQ ID NO:23 or its substantial equivalent thereto;-   (29) a partial peptide of any of the G protein-coupled receptor    proteins described in (25)-(28) above or a salt thereof;-   (30) a DNA which comprises a DNA having a nucleotide sequence coding    for the G protein-coupled receptor protein described in (25) above;-   (31) a DNA which comprises a DNA having a nucleotide sequence coding    for the G protein-coupled receptor protein described in (26) above;-   (32) a DNA which comprises a DNA having a nucleotide sequence coding    for the G protein-coupled receptor protein described in (27) above;-   (33) a DNA which comprises a DNA having a nucleotide sequence coding    for the G protein-coupled receptor protein described in (28) above;-   (34) the DNA described in (30) above, which comprises the nucleotide    sequence of SEQ ID NO:24 or the nucleotide sequence of SEQ ID NO:25;-   (35) the DNA described in (31) above, which comprises the nucleotide    sequence of SEQ ID NO:26;-   (36) the DNA described in (32) above, which comprises the nucleotide    sequence of SEQ ID NO:27;-   (37) the DNA described in (33) above, which comprises the nucleotide    sequence of SEQ ID NO:28;-   (38) a recombinant vector comprising any of the DNAs described in    (30)-(33) above;-   (39) a transformant carrying the recombinant vector described    in (38) above;-   (40) a method for producing the G protein-coupled receptor protein    described in (28) above or a salt thereof, which comprises culturing    the transformant of (39) to produce said G protein-coupled receptor    protein on the cell membrane of the transformant;-   (41) an antibody to any of the G protein-coupled receptor protein    described in (25)-(28) above or a salt thereof, or the partial    peptide described in (29) above or a salt thereof.

To be further specific, the G protein-coupled receptor protein relatesto:

-   (42) the G protein-coupled receptor protein described in (25) above    or a salt thereof, wherein the protein comprises (i) an amino acid    sequence selected from the group consisting of an amino acid    sequence of SEQ ID NO:19, amino acid sequences wherein 1 to 30 amino    acid residues, preferably 1 to 10 amino acid residues are deleted    from the amino acid sequence of SEQ ID NO:19, amino acid sequences    wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid    residues are added to the amino acid sequence of SEQ ID NO:19, and    amino acid sequences wherein 1 to 0.30 amino acid residues,    preferably 1 to 10 amino acid residues in the amino acid sequence of    SEQ ID NO:19 are substituted with one or more than amino acid    residues and/or (ii) an amino acid sequence selected from the group    consisting of an amino acid sequence of SEQ ID NO:20, amino acid    sequences wherein 1 to 30 amino acid residues, preferably 1 to 10    amino acid residues are deleted from the amino acid sequence of SEQ    ID NO:20, amino acid sequences wherein 1 to 30 amino acid residues,    preferably 1 to 10 amino acid residues are added to the amino acid    sequence of SEQ ID NO:20, and amino acid sequences wherein 1 to 30    amino acid residues, preferably 1 to 10 amino acid residues in the    amino acid sequence of SEQ ID NO:20 are substituted with one or more    other amino acid residues;-   (43) the G protein-coupled receptor protein described in (26) above    or a salt thereof, wherein the protein comprises an amino acid    sequence selected from the group consisting of an amino acid    sequence of SEQ ID NO:21, amino acid sequences wherein 1 to 30 amino    acid residues, preferably 1 to 10 amino acid residues are deleted    from the amino acid sequence of SEQ ID NO:21, amino acid sequences    wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid    residues are added to the amino acid sequence of SEQ ID NO:21, and    amino acid sequences wherein 1 to 30 amino acid residues, preferably    1 to 10 amino acid residues in the amino acid sequence of SEQ ID    NO:21 are substituted with one or more other amino acid residues;-   (44) the G protein-coupled receptor protein described in (27) above    or a salt thereof wherein the protein comprises an amino acid    sequence selected from the group consisting of an amino acid    sequence of SEQ ID NO:22, amino acid sequences wherein 1 to 30 amino    acid residues, preferably 1 to 10 amino acid residues are deleted    from the amino acid sequence of SEQ ID NO:22, amino acid sequences    wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid    residues are added to the amino acid sequence of SEQ ID NO:22, and    amino acid sequences wherein 1 to 30 amino acid residues, preferably    1 to 10 amino acid residues in the amino acid sequence of SEQ ID    NO:22 are substituted with one or more other amino acid residues;-   (45) the G protein-coupled receptor protein described in (28) above    or a salt thereof, wherein the protein comprises an amino acid    sequence selected from the group consisting of an amino acid    sequence of SEQ ID NO:23, amino acid sequences wherein 1 to 30 amino    acid residues, preferably 1 to 10 amino acid residues are deleted    from the amino acid sequence of SEQ ID NO:23, amino acid sequences    wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid    residues are added to the amino acid sequence of SEQ ID NO:23, and    amino acid sequences wherein 1 to 30 amino acid residues, preferably    1 to 10 amino acid residues in the amino acid sequence of SEQ ID    NO:23 are substituted with one or more other amino acid residues.

As used herein the term “substantial equivalent(s)” means that theactivity of the protein, e.g., nature of the binding activity of theligand and the receptor and physical characteristics are substantiallythe same. Substitutions, deletions or insertions of amino acids often donot produce radical changes in the physical and chemical characteristicsof a polypeptide, in which case polypeptides containing thesubstitution, deletion, or insertion would be considered to besubstantially equivalent to polypeptides lacking the substitution,deletion, or insertion. Substantially equivalent substitutes for anamino acid within the sequence may be selected from other members of theclass to which the amino acid belongs. The non-polar (hydrophobic) aminoacids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of the human pituitary-derived Gprotein-coupled receptor protein cDNA fragment harbored in cDNA clonep19P2 isolated by PCR using human pituitary-derived cDNA and the aminoacid encoded by the nucleotide sequence. The primer used for sequencingwas −21M13. The underscored region correspond to the synthetic primer.

FIG. 2 shows the nucleotide sequence of the human pituitary-derived Gprotein-coupled receptor protein cDNA fragment harbored in cDNA clonep19P2 isolated by PCR using human pituitary-derived cDNA and the aminoacid sequence encoded thereby. The primer used for sequencing wasM13RV-N (Takara). The underscored region correspond to the syntheticprimer.

FIG. 3 shows a partial hydrophobic plot of the protein encoded by thehuman pituitary-derived G protein-coupled receptor protein cDNA fragmentharbored in p19P2 constructed according to the amino acid sequence shownin FIG. 1.

FIG. 4 shows a partial hydrophobic plot of the protein encoded by thehuman pituitary-derived G protein-coupled receptor protein cDNA fragmentharbored in p19P2 constructed according to the amino acid sequence shownin FIG. 2.

FIG. 5 is a diagram comparing the partial amino acid sequence of theprotein encoded by the human pituitary-derived G protein-coupledreceptor protein cDNA fragment harbored in p19P2 as shown in FIGS. 1 and2 with the known G protein-coupled receptor protein S12863. The shadowedregion represents the region of agreement. The 1 to 9 amino acidsequence of p19P2 corresponds to the 1 to 99 amino acid sequence of FIG.1 and the 156 to 230 amino acid sequence corresponds to the 1 to 68amino acid sequence of FIG. 2.

FIG. 6 shows the nucleotide sequence of the MIN6-derived Gprotein-coupled receptor protein cDNA fragment based on the nucleotidesequences of the MIN6-derived G protein-coupled receptor protein cDNAfragments harbored in the cDNA clones pG3-2 and pG1-10 isolated by PCRusing MIN6-derived cDNA and the amino acid sequence encoded by thenucleotide sequence. The underscored region correspond to the syntheticprimer.

FIG. 7 is a diagram comparing the partial amino acid sequence encoded bypG3-2/pG1-10 of the MIN6-derived G protein-coupled receptor proteinshown in FIG. 6 with the partial amino acid sequence of the proteinencoded by p19P2 shown in FIGS. 1 and 2. The shadowed region correspondsto the region of agreement. The 1 to 99 amino acid sequence of theprotein encoded by p19P2 corresponds to the 1 to 99 amino acid sequenceof FIG. 1 and the 156 to 223 amino acid sequence corresponds to the 1 to68 amino acid sequence of FIG. 2. The 1 to 223 amino acid sequence ofthe protein encoded by pG3-2/pG1-10 corresponds to the 1 to 223 aminoacid sequence of FIG. 6.

FIG. 8 is a partial hydrophobic plot of the MIN6-derived Gprotein-coupled receptor protein constructed according to the partialamino acid sequence shown in FIG. 6.

FIG. 9 shows the entire nucleotide sequence of the humanpituitary-derived G protein-coupled receptor protein cDNA harbored inthe cDNA clone phGR3 isolated from a human pituitary-derived cDNAlibrary by the plaque hybridization method using the DNA fragmentinserted in p19P2 as a probe and the amino acid sequence encorded by thenucleotide sequence.

FIG. 10 shows the result of Northern blotting of human pituitary mRNAhybridized with radioisotope-labeled human pituitary cDNA clone phGR3.

FIG. 11 shows a hydrophobic plot of the protein encoded by the humanpituitary-derived G protein-coupled receptor protein cDNA harbored inthe phGR3 as constructed according to the amino acid sequence shown inFIG. 9.

FIG. 12 shows the nucleotide sequence of the MIN6-derived Gprotein-coupled receptor protein cDNA fragment harbored in the cDNAclone p5S38 isolated by PCR using MIN6-derived cDNA and the amino acidsequence encoded by the nucleotide sequence. The underscored regioncorrespond to the synthetic primer.

FIG. 13 shows a diagram comparing the partial amino acid sequence ofMIN6-derived G protein-coupled receptor protein encoded by p5S38 shownin FIG. 12 with the partial amino acid sequence of G protein-coupledreceptor protein encoded by the cDNA fragment harbored in p19P2 as shownin FIGS. 1 and 2 and the partial amino acid sequence of Gprotein-coupled receptor protein encoded by the nucleotide sequencegenerated from the nucleotide sequences of cDNA fragments contained inpG3-2 and pG1-10 shown in FIG. 6. The shadowed region represents thesequence region of agreement. The 1 to 144 amino acid sequence of theprotein encoded by p5S38 corresponds to the 1 to 144 amino acid sequenceof FIG. 12, the 1 to 99 amino acid sequence of the protein encoded byp19P2 corresponds to the 1 to 99 amino acid sequence of FIG. 1 and the156 to 223 amino acid sequence corresponds to 1 to 68 amino acidsequence of FIG. 2. The 1 to 223 amino acid sequence of the proteinencoded by pG3-2/pG1-10 corresponds to the 1 to 223 amino acid sequenceof FIG. 6.

FIG. 14 shows a partial hydrophobic plot of the protein encoded by theMIN6-derived G protein-coupled receptor protein cDNA harbored in p5S38as constructed according to the partial amino acid sequence shown inFIG. 12.

FIG. 15 shows the results of the following analysis. Thus, RT-PCR wascarried out to confirm the expression of mRNA in CHO cells transfectedby pAKKO-19P2. Lanes 1-7 represent the results obtained by performingPCRs using serial dilutions of pAKKO-19P2 for comparison, i.e. the 10μl/ml stock solution (lane 1), 1/2 dilution (lane 2), 1/4 dilution (lane3), 1/64 dilution (Lane 4), 1/256 dilution (lane 5), 1/1024 dilution(lane 6), and 1/4096 dilution (lane 7) of the plasmid as templates, andanalyzing the reaction mixtures by 1.2% agarose gel electrophoresis.Lanes 8 through 11 are the results obtained by performing PCRs using a1/10 dilution (lane 8), a 1/100 dilution (lane 9), and a 1/1000 dilution(lane 10) of the cDNA prepared from the CHO-19P2 cell line as templatesand subjecting the respective reaction mixtures to electrophoresis. Lane11 was obtained by performing PCR using a template obtained by carryingout cDNA synthesis without reverse transcriptase and subjecting the PCRreaction product to electrophoresis. Lanes 12 and 13 were obtained byperforming PCR using cDNAs prepared from mock CHO cells with and withoutaddition of reverse transcriptase, respectively, as templates andsubjecting the respective reaction products to electrophoresis. Mrepresents the DNA size marker. The lanes at both ends were obtained byelectrophoresing 1 μl of λ/Sty I digest (Nippon Gene) and the secondlane from right was obtained with 1 μl of φ/χ174/Hinc II digest (NipponGene). The arrowmark indicates the position of the band amplified by PCRof about 400 bp.

FIG. 16 shows the activity of the crude ligand peptide fractionextracted from rat whole brain to promote release of arachidonic acidmetabolites from CHO-19P2 cells. The arachidonic acid metabolitereleasing activity was expressed as % of the amount of [³H] arachidonicacid metabolites released in the presence of the crude ligandpolypeptide fraction with the amount of [³H] arachidonic acidmetabolites released in the presence of 0.05% BAS-HABB being taken as100%. The activity to promote release of arachidonic acid metabolitesfrom the CHO-19P2 cell line was detected in a 30% CH₃CN fraction.

FIG. 17 shows the activity of the crude ligand polypeptide fractionextracted from bovine hypothalamus to promote release of arachidonicacid metabolites from CHO-19P2 cells. The arachidonic acid metaboliterelease-promoting activity was expressed as % of the amount of [³H]arachidonic acid metabolites released in the presence of the crudeligand polypeptide fraction with the amount of [³H] arachidonic acidmetabolites released in the presence of 0.05% BAS-HABB being taken as100%. The activity to promote release of arachidonic acid metabolitesfrom the CHO-19P2 cell line was detected in a 30% CH₃CN fraction just asin the crude ligand polypeptide fraction from rat whole brain.

FIG. 18 shows the activity of the fraction purified with thereversed-phase column C18 218TP5415 to specifically promote release ofarachidonic acid metabolites from CHO-19P2 cells. The active fractionfrom RESOURCE S was fractionated on C18 218TP5415. Thus, chromatographywas carried out at a flow rate of 1 ml/min. on a concentration gradientof 20%-30% CH₃CN/0.1% TFA/H₂O, the eluate was collected in 1 mlfractions, and each fraction was lyophilized. Then, the activity of eachfraction to specifically promote release of arachidonic acid metabolitesfrom the CHO-19P2 cell line was determined. As a result, the activitywas fractionated into 3 fractions (designated, in the order of elution,as P-1, P-2, and P-3).

FIG. 19 shows the activity of the fraction purified with thereversed-phase column diphenyl 219TP5415 to specifically promotearachidonic acid metabolite release from CHO-19P2 cells. The P-3 activefraction from C18 218TP5415 was fractionated on diphenyl 219TP5415. Thechromatography was carried out at a flow rate of 1 ml/min. on aconcentration gradient of 22%-25% CH₃CN/0.1% TFA/H₂O, the eluate wascollected in 1 ml fractions, and each fraction was lyophilized. Then,the activity to specifically promote release of arachidonic acidmetabolites from CHO-19P2 cells in each fraction was determined. As aresult, the activity converged in a single peak.

FIG. 20 shows the activity of the fraction purified by reversed-phasecolumn μRPC C2/C18 SC 2.1/10 to specifically promote release ofarachidonic acid metabolites from CHO-19P2 cells. The peak activefraction from diphenyl 219TP5415 was fractionated on μRPC C2/C18 SC2.1/10. The chromatography was carried out at a flow rate of 100 μl/min.on a concentration gradient of 22%-23.5% CH₃CN/0.1% TFA/H₂O, the eluatewas collected in 100 μl fractions, and each fraction was lyophilized.Then, the activity to specifically promote release of arachidonic acidmetabolites from CHO-19P2 cells in each fraction was determined. As aresult, the activity was found as two peaks of apparently a singlesubstance (peptide).

FIG. 21 shows the activity of the P-2 fraction purified byreversed-phase column μRPC C2/C18 SC 2.1/10 to specifically promoterelease of arachidonic acid metabolites from CHO-19P2 cells. Thechromatography was carried out at a flow rate of 100 μl/min. on aconcentration gradient of 21.5%-23.0% CH₃CN/0.1% TFA/dH₂O, the eluatewas collected in 100 μl fractions, and each fraction was lyophilized.Then, the activity to specifically promote release of arachidonic acidmetabolites from CHO-19P2 cells in each fraction was determined. As aresult, the activity was found as a peak of apparently a singlesubstance.

FIG. 22 shows the nucleotide sequence of bovine hypothalamus ligandpolypeptide cDNA fragment as derived from the nucleotide sequence of thebovine hypothalamus-derived ligand polypeptide cDNA fragment whichspecifically promotes release of arachidonic acid metabolites fromCHO-19P2 cells as harbored in a cDNA clone isolated by PCR using bovinehypothalamus-derived cDNA and the amino acid sequence encoded by saidnucleotide sequence. The region indicated by the arrowmark correspondsto the synthetic primer.

FIG. 23 shows the nucleotide sequence of the bovine hypothalamus-derivedligand polypeptide cDNA fragment generated according to the nucleotidesequence of the bovine hypothalamus-derived ligand polypeptide cDNAfragment which specifically promotes release of arachidonic acidmetabolites from CHO-19P2 cells as harbored in a cDNA clone isolated byPCR using bovine hypothalamus-derived cDNA and the amino acid sequenceencoded by said nucleotide sequence. The region indicated by thearrowmark corresponds to the synthetic primer.

FIG. 24 shows the amino acid sequences (a) and (b) of the bovinehypothalamus-derived ligand polypeptides which specifically promoterelease of arachidonic acid metabolites from CHO-19P2 cells and the cDNAsequence coding for the full coding region of the ligand polypeptidesdefined by SEQ ID NO:1 and SEQ ID NO:44.

FIG. 25 shows the concentration-dependent activity of synthetic ligandpolypeptide (19P2-L31) to specifically promote release of arachidonicacid metabolites from CHO-19P2 cells. The synthetic peptide wasdissolved in degassed dH₂O at a final concentration of 10⁻³M and dilutedwith 0.05% BSA-HBSS to concentrations of 10⁻¹²M-10⁻⁶M. The arachidonicacid metabolite releasing activity was expressed in the measuredradioactivity of [³H] arachidonic acid metabolites released in thesupernatant when the dilution was added to the cells. As a result, theactivity of 19P2-31 to specifically promote release of arachidonic acidmetabolites from CHO-19P2 cells was found in a concentration-dependentmanner.

FIG. 26 shows the concentration-dependent activity of synthetic ligandpolypeptide (19P2-L31(O)) to specifically promote release of arachidonicacid metabolites from CHO-19P2 cells. The synthetic ligand peptide wasdissolved in degassed dH₂O at a final concentration of 10⁻³ M anddiluted with 0.05% BSA-HBSS to concentrations of 10⁻¹²M-10⁻⁶M. Thearachidonic acid metabolite releasing activity was expressed in themeasured radioactivity of [³H] arachidonic acid metabolites released inthe supernatant when the dilution was added to the cells. As a result,the activity of 19P2-L31(O) to specifically promote release ofarachidonic acid metabolites from CHO-19P2 cells was found in adose-dependent manner.

FIG. 27 shows the activity of synthetic ligand polypeptide 19P2-L20 tospecifically promote release of arachidonic acid metabolites fromCHO-19P2 cells. The synthetic peptide was dissolved in degassed dH₂O ata final concentration of 10⁻³M and diluted with 0.05% BSA-HBSS toconcentrations of 10 ¹²M-10⁻⁶M. The arachidonic acid metabolitereleasing activity was expressed in the measured radioactivity of [³H]arachidonic acid metabolites released in the supernatant when thedilution was added to the cells. As a result, the activity of 19P2-L20to specifically promote release of arachidonic acid metabolites fromCHO-19P2 cells was found in a dose-dependent manner.

FIG. 28 shows the 1.2% agarose gel electrophoregram of the DNA fragmentsof the phages cloned from a bovine genomic library as digested withrestriction enzymes BamHI(B) and SalI(S). As the DNA size marker (M),StyI digests of λ phage DNA were used. In lane B, two bands derived fromthe vector were detected in positions between the first (19,329 bp) andsecond (7.743 bp) marker bands, as well as 3 bands derived from theinserted fragment between the third (6,223 bp) and 5th (3,472 bp) bands.In lane S, two bands derived from the vector were similarly detected butdue to the overlap of the band of the inserted fragment, the upper bandis thicker than the band in lane B.

FIG. 29 shows the nucleotide sequence around the coding region asdecoded from bovine genomic DNA. The 1st to 3rd bases (ATG) correspondto the translation start codon and the 767th to 769th bases (TAA)correspond to the translation end codon.

FIG. 30 shows a comparison between the nucleotide sequence (genome)around the coding region as deduced from bovine genomic DNA and thenucleotide sequence (cDNA) of bovine cDNA cloned by PCR. The sequenceregion of agreement is indicated by shading. As to the 101st to 572ndregion, there is no corresponding region in the nucleotide sequence ofcDNA, indicating that it is an intron.

FIG. 31 shows the translation of the amino acid sequence encoded afterelimination of the intron from the nucleotide sequence around the codingregion as decoded from bovine genomic DNA.

FIG. 32 shows the full-length amino acid sequence and the cDNA sequencecoding for the full coding region of rat ligand polypeptide.

FIG. 33 shows amino acid sequence of bovine ligand polypeptide and thenucleotide sequences of DNAs coding for bovine polypeptide and ratpolypeptide. The arrowmark indicates the region corresponding to thesynthetic primer.

FIG. 34 shows the full-length amino acid sequence and the sequence ofcDNA coding for the full coding region of human ligand polypeptide.

FIG. 35 shows a comparison of the amino acid sequences in thetranslation region of bovine ligand polypeptide, rat ligand polypeptide,and human ligand polypeptide.

FIG. 36 shows the results of receptor binding experiments on livingcells wherein radioiodinated ligand polypeptide is used in theexperiments.

FIG. 37 shows the results of measurements of release of arachidonic acidmetabolites from CHO-19P2-9 and CHO-UHR1 by ligand polypeptide.

FIG. 38 shows the results of quantification of UHR-1 mRNA by RT-PCR indiscrete regions of the brain and tissues in rats.

FIG. 39 shows the results of quantification of ligand polypeptide mRNAby RT-PCR in discrete regions of the brain and tissues in rats.

FIG. 40 shows effects of ligand polypeptide on glucose-induced increasein plasma insulin concentration, which is measured by radioimmunoassay.

FIG. 41 shows the results of measurements of motor activity byadministration of 10 nmol of ligand polypeptide to mouse.

(a) relates to spontaneous motor activity and (b) relates to rearing.

FIG. 42 shows the results of measurements of motor activity byadministration of 1 nmol of ligand polypeptide to mouse.

(a) relates to spontaneous motor activity and (b) relates to rearing.

FIG. 43 shows the results of measurements of motor activity byadministration of 0.1 nmol of ligand polypeptide to mouse.

(a) relates to spontaneous motor activity and (b) relates to rearing.

FIG. 44 shows the results of measurements of motor activity byadministration of 0.01 mmol of ligand polypeptide to mouse.

(a) relates to spontaneous motor activity and (b) relates to rearing.

FIG. 45 shows the results of measurements of body temperature which ismeasured at the time when the ligand polypeptide is administered to thelateral ventricle of mice. The administration of ligand polypeptide iscarried out after 15 hours from administration of reserpine at a dose of3 mg/kg, S.C.

In FIG. 45, the single star mark asterisk shows p<0.05 and the doublestar marks asterisks shows p<0.01.

FIG. 46 illustrates the drawing in which the micro-injection cannula isinserted into the area postrema at an angle of 20°.

FIG. 47 shows the typical example of direct and average blood pressurewhich is measured after the injection of ligand polypeptide into thearea postrema of rat. It is measured after the injection of 10 nmol ofligand polypeptide at the rate of 1 μl/min, and under the condition ofnon-anesthesia.

FIG. 48 shows the results of measurements of growth hormone (GH) inplasma when 50 nmol of ligand polypeptide is administered into the thirdventricle of rat after anesthesia by pentobarbital.

FIG. 49 shows the changes of secretion of GH in plasma by administrationof 50 nmol of ligand polypeptide into the third ventricle in freelymoving rats.

The ligand polypeptide or PBS was administered into the third ventricle.At 10 min later, 5 μg/kg of GHRH was administered intravenously to therat conscious. GH levels were measured just prior to intraventricularadministration (time 0) and 10, 20, 30, 40, and 60 min after theintravenous injection of GHRH.

In FIG. 49, the single star mark asterisk shows p<0.05 and the doublestar marks asterisks show p<0.01.

FIG. 50 shows the relationship between the ligand polypeptide serum andthe absorbance.

FIG. 51 shows the inhibition of the release of archidonic acidmetabolites by anti-ligand polypeptide polyclonal antibody.

FIG. 52 shows the sequence of cDNA coding for UHR-1, which isconstructed on pAKKO-UHR1-7.

BEST MODE FOR CARRYING OUT THE INVENTION

The ligand polypeptide according to the present invention is apolypeptide which is capable of binding to G protein-coupled receptorprotein and comprising an amino acid sequence represented by SEQ IDNO:73 or its substantial equivalent thereto or a partial peptidethereof, or its amide or ester, or a salt thereof. In SEQ ID NO:73, Xaaat 10th position is Ala or Thr; Xaa at 11th position is Gly or Ser; andXaa at 21th position is H, Gly, or GlyArg.

The above ligand polypeptide, its amide or ester, or a salt thereof(hereinafter sometimes referred to briefly as the ligand polypeptide orthe polypeptide), processes for their production, and uses for thepolypeptide are now described in detail.

The above ligand polypeptide of the present invention includes anypolypeptides derived from any tissues, e.g. pituitary gland, pancreas,brain, kidney, liver, gonad, thyroid gland, gall bladder, bone marrow,adrenal gland, skin, muscle, lung, digestive canal, blood vessel, heart,etc.; or cells of man and other warm-blooded animals, e.g. guinea pig,rat, mouse, swine, sheep, bovine, monkey, etc. and comprising an aminoacid sequence represented by SEQ ID NO:73 or its substantial equivalentthereto. For example, in addition to the protein comprising the aminoacid sequence of SEQ ID NO:73, the ligand polypeptide of the presentinvention includes the protein comprising an amino acid sequence havinga homology of about 50-99.9%, preferably 70-99.9%, more preferably80-99.9% and especially preferably 90-99.9% to the amino acid sequenceof SEQ ID NO:73 and having qualitatively substantially equivalentactivity to the protein comprising the amino acid sequence of SEQ IDNO:73. The term “substantially equivalent” means the nature of thereceptor-binding activity, signal transduction activity and the like isequivalent. Thus, it is allowable that even differences among gradessuch as the strength of receptor binding activity and the molecularweight of the polypeptide are present.

To be more specific, the ligand polypeptide of the present inventionincludes the polypeptide derived from the rat whole brain, bovinehypothalamus, or human whole brain and comprising the amino acidsequence of SEQ ID NO:73. In addition, the ligand polypeptide of thepresent invention includes the polypeptides which comprises substantialequivalent polypeptides such as polypeptides wherein 1 to 15, preferably1 to 10, and more preferably 1 to 5 amino acid residues are deleted fromthe amino acid sequence of SEQ ID NO:73, polypeptides wherein 1 to 80,preferably 1 to 50, more preferably 1 to 10 amino acid residues areadded to the amino acid sequence of SEQ ID NO:73, or polypeptideswherein 1 to 15, preferably 1 to 10, more preferably 1 to 5 amino acidresidues are substituted with one or more other amino acid residues.

The amino acid sequence of SEQ ID NO:73 comprises SEQ ID NO:8, 9, 10,50, 51, 52, 64, 65 or 66. The substantial equivalent polypeptides to thepolypeptide comprising the amino acid sequence of SEQ ID NO: 73 arepolypeptides comprising the amino acid sequences of SEQ ID NO:1, 3, 4,5, 6, 7, 44, 45, 47, 48, 49, 59, 61, 62, or 63.

Among them, preferred is the polypeptide comprising the amino acidsequence of SEQ ID NO:73 and the polypeptide comprising the amino acidsequence which a peptide of SEQ ID NO:74 is added to the N-terminus ofthe polypeptide comprising the amino acid sequence of SEQ ID NO:73.

Furthermore, the polypeptide or partial peptide of the present inventionincludes those wherein the N-terminal side of Gln is cleaved in vivo toform pyroglutamyl peptide.

The peptides described in this specification, the left ends are theN-terminus (amino terminus) and the right end is the C-terminus(carboxyl terminus) according to the convention of the peptideindication. While the C-terminus of the polypeptide of SEQ ID NO:73 isusually carboxyl (—COOH) or carboxylate (—COO⁻), it may be amide(—CONH₂) or ester (—COOR) form. The ester residue R includes a C₁₋₆alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc., aC₃₋₈ cycloalkyl group such as cyclopentyl, cyclohexyl, etc., a C₆₋₁₂aryl group such as phenyl, α-naphthyl, etc., and a C₇₋₁₄ aralkyl groupsuch as a phenyl-C₁₋₂ alkyl group, e.g. benzyl, phenethyl, benzhydryl,etc. or an α-naphthyl-C₁₋₂ alkyl, e.g. α-naphthylmethyl etc. Inaddition, the ester may be a pivaloyloxymethyl ester which is broadlyused for oral administration. When the polypeptide of SEQ ID NO:73 has acarboxyl or carboxylate group in any position other than the C-terminus,the corresponding amide or ester are also included in the concept of thepolypeptide of the present invention. The ester mentioned just aboveincludes the esters mentioned for the C-terminus.

The preferred ligand polypeptide of the present invention is a peptidewhich the C-terminus is amidated. Especially preferred is a polypeptidecomprising the amino acid sequence of SEQ ID NO:5, 8, 47, 50, 61 or 64which the C-terminus is amidated.

The salt of polypeptide of the present invention includes salts withphysiologically acceptable bases, e.g. alkali metals or acids such asorganic or inorganic acids, and is preferably a physiologicallyacceptable acid addition salt. Examples of such salts are salts thereofwith inorganic acids, e.g. hydrochloric acid, phosphoric acid,hydrobromic acid or sulfuric acid, etc. and salts thereof with organicacids, e.g. acetic acid, formic acid, propionic acid, fumaric acid,maleic acid, succinic acid, tartaric acid, citric acid, malic acid,oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid,etc.

The ligand polypeptide or amide or ester, or a salt thereof of thepresent invention may be manufactured from the tissues or cells ofwarm-blooded animals inclusive of human by purifying techniques ormanufactured by the peptide synthesis as described hereinafter.Moreover, it can be manufactured by culturing a transformant carrying aDNA coding for the polypeptide as described hereinafter.

In the production from the tissues or cells of human or otherwarm-blooded animals, the ligand polypeptide can be purified andisolated by a process which comprises homogenizing the tissue or cellsof human or other warm-blooded animal, extracting the homogenate with anacid, for instance, and subjecting the extract to a combination ofchromatographic procedures such as reversed-phase chromatography,ion-exchange chromatography, affinity chromatography, etc.

As mentioned above, the ligand polypeptide in the present invention canbe produced by the per se known procedures for peptide synthesis. Themethods for peptide synthesis may be any of a solid-phase synthesis anda liquid-phase synthesis. Thus, the objective peptide can be produced bycondensing a partial peptide or amino acid capable of constituting theprotein with the residual part thereof and, when the product has aprotective group, the protective group is detached whereupon a desiredpeptide can be manufactured. The known methods for condensation anddeprotection includes the procedures described in the followingliterature (1)-(5).

-   (1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience    Publishers, New York, 1966-   (2) Schroeder and Luebke, The Peptide, Academic Press, New York,    1965-   (3) Nobuo Izumiya et al., Fundamentals and Experiments in Peptide    Synthesis, Maruzen, 1975-   (4) Haruaki Yajima and Shumpei Sakakibara, Biochemical Experiment    Series 1, Protein Chemistry IV, 205, 1977-   (5) Haruaki Yajima (ed.), Development of Drugs-Continued, 14,    Peptide Synthesis, Hirokawa Shoten

After the reaction, the protein can be purified and isolated by acombination of conventional purification techniques such as solventextraction, column chromatography, liquid chromatography, andrecrystallization. Where the protein isolated as above is a freecompound, it can be converted to a suitable salt by the known method.Conversely where the isolated product is a salt, it can be converted tothe free peptide by the known method.

The amide of polypeptide can be obtained by using a resin for peptidesynthesis which is suited for amidation. The resin includes chloromethylresin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAMresin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamideresin, 4-(2′,4′-dimethoxyphenylhydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenylFmoc aminoethyl)phenoxy resin, and so on. Usingsuch a resin, amino acids whose α-amino groups and functional groups ofside-chain have been suitably protected are condensed on the resinaccording to the sequence of the objective peptide by variouscondensation techniques which are known per se. At the end of the seriesof reactions, the peptide or the protected peptide is removed from theresin and the protective groups are removed to obtain the objectivepolypeptide.

For the condensation of the above-mentioned protected amino acids, avariety of activating reagents for peptide synthesis can be used but acarbodiimide compound is particularly suitable. The carbodiimideincludes DCC, N,N′-diisopropylcarbodiimide, andN-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide. For activation with sucha reagent, a racemization inhibitor additive, e.g. HOBt and theprotected amino acid are directly added to the resin or the protectedamino acid pre-activated as symmetric acid anhydride, HOBt ester, orHOOBt ester is added to the resin. The solvent for the activation ofprotected amino acids or condensation with the resin can be properlyselected from among those solvents which are known to be useful forpeptide condensation reactions. For example, N,N-dimethylformamide,N-methylpyrrolidone, chloroform, trifluoroethanol, dimethyl sulfoxide,DMF, pyridine, dioxane, methylene chloride, tetrahydrofuran,acetonitrile, ethyl acetate, or suitable mixtures of them can bementioned. The reaction temperature can be selected from the rangehitherto-known to be useful for peptide bond formation and is usuallyselected from the range of about −20° C.-50° C. The activated amino acidderivative is generally used in a proportion of 1.5-4 fold excess. Ifthe condensation is found to be insufficient by a test utilizing theninhydrin reaction, the condensation reaction can be repeated to achievea sufficient condensation without removing the protective group. Ifrepeated condensation still fails to provide a sufficient degree ofcondensation, the unreacted amino group can be acetylated with aceticanhydride or acetylimidazole.

The protecting group of amino group for the starting material amino acidincludes Z, Boc, tertiaryamyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl,trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, or Fmoc. The carboxy-protecting group that canbe used includes but is not limited to the above-mentioned C₁₋₆ alkyl,C₃₋₈ cycloalkyl and C₇₋₁₄ aralkyl as well as 2-adamantyl, 4-nitrobenzyl,4-methoxybenzyl, 4-chlorobenzyl, phenacyl, benzyloxycarbonylhydrazido,tertiary-butoxycarbonylhydrazido, and tritylhydrazido.

The hydroxy group of serine and threonine can be protected byesterification or etherification. The group suited for saidesterification includes carbon-derived groups such as lower alkanoylgroups, e.g. acetyl etc., aroyl groups, e.g. benzoyl etc.,benzyloxycarbonyl, and ethoxycarbonyl. The group suited for saidetherification includes benzyl, tetrahydropyranyl, and tertiary-butyl.

The protective group for the phenolic hydroxyl group of tyrosineincludes Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, and tertiary-butyl.

The protecting group of imidazole for histidine includes Tos,4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum,Boc, Trt, and Fmoc.

The activated carboxyl group of the starting amino acid includes thecorresponding acid anhydride, azide, and active esters, e.g. esters withalcohols such as pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt, etc. The activatedamino group of the starting amino acid includes the correspondingphosphoramide.

The method for elimination of protective groups includes catalyticreduction using hydrogen gas in the presence of a catalyst such aspalladium black or palladium-on-carbon, acid treatment with anhydroushydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid,trifluoroacetic acid, or a mixture of such acids, base treatment withdiisopropylethylamine, triethylamine, piperidine, piperazine, reductionwith sodium metal in liquid ammonia. The elimination reaction by theabove-mentioned acid treatment is generally carried out at a temperatureof −20° C.-40° C. and can be conducted advantageously with addition of acation acceptor such as anisole, phenol, thioanisole, m-cresol,p-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol. The2,4-dinitrophenyl group used for protecting the imidazole group ofhistidine can be eliminated by treatment with thiophenol, while theformyl group used for protecting the indole group of tryptophan can beeliminated by alkali treatment with dilute sodium hydroxide solution ordilute aqueous ammonia as well as the above-mentioned acid treatment inthe presence of 1,2-ethanedithiol, 1,4-butanedithiol.

The method for protecting functional groups which should not take partin the reaction of the starting material, the protective groups that canbe used, the method of removing the protective groups, and the method ofactivating the functional groups that are to take part in the reactioncan all be selected judicially from among the known groups and methods.

An another method for obtaining the amide form of the polypeptidecomprises amidating the α-carboxyl group of the C-terminal amino acid atfirst, then extending the peptide chain to the N-side until the desiredchain length, and then selectively deprotecting the α-amino group of theC-terminal peptide and the α-carboxy group of the amino acid or peptidethat is to form the remainder of the objective polypeptide andcondensing the two fragments whose α-amino group and side-chainfunctional groups have been protected with suitable protective groupsmentioned above in a mixed solvent such as that mentioned hereinbefore.The parameters of this condensation reaction can be the same asdescribed hereinbefore. From the protected peptide obtained bycondensation, all the protective groups are removed by theabove-described method to thereby provide the desired crude peptide.This crude peptide can be purified by known purification procedures andthe main fraction be lyophilized to provide the objective amidatedpolypeptide.

To obtain an ester of the polypeptide, the α-carboxyl group of theC-terminal amino acid is condensed with a desired alcohol to give anamino acid ester and then, the procedure described above for productionof the amide is followed.

The partial peptide of the ligand polypeptide of the present invention,its amide or ester, or a salt thereof can be any peptide that has thesame activities, e.g. pituitary function modulating activity, centralnervous system function modulating activity, or pancreatic functionmodulating activity as the polypeptide which has an amino acid sequenceof SEQ ID NO:73 or its substantial equivalent thereto. As such peptides,there can be mentioned peptides wherein 1 to 15 amino acids residues aredeleted from the above-mentioned amino acid sequence of SEQ ID NO:73. Tobe specific, the peptide having an amino acid sequence corresponding tothe 2nd to 21st positions of the amino acid sequence of SEQ ID NO:73,the peptide corresponding to the 3rd to 21st positions of the amino acidsequence of SEQ ID NO:73, the peptide corresponding to the 4th to 21stpositions of the amino acid sequence of SEQ ID NO:73, the peptidecorresponding to the 5th to 21st positions of the amino acid sequence ofSEQ ID NO:73, the peptide corresponding to the 6th to 21st positions ofthe amino acid sequence of SEQ ID NO:73, the peptide corresponding tothe 7th to 21st positions of the amino acid sequence of SEQ ID NO:73,the peptide corresponding to the 8th to 21st positions of the amino acidsequence of SEQ ID NO:73, the peptide corresponding to the 9th to 21stpositions of the amino acid sequence of SEQ ID NO:73, the peptidecorresponding to the 10th to 21st positions of the amino acid sequenceof SEQ ID NO:73, the peptide corresponding to the 11th to 21st positionsof the amino acid sequence of SEQ ID NO:73, the peptide corresponding tothe 12th to 21st positions of the amino acid sequence of SEQ ID NO:73,the peptide corresponding to the 13th to 21st positions of the aminoacid sequence of SEQ ID NO:73, the peptide corresponding to the 14th to21st positions of the amino acid sequence of SEQ ID NO:73, and thepeptide corresponding to the 15th to 21st positions of the amino acidsequence of SEQ ID NO:73, can be mentioned as preferred examples.Moreover, the peptide having the amino acid sequence of SEQ ID NO:74 isalso preferred.

The ligand polypeptide or partial peptide thereof can be used as antigenfor preparation of anti-ligand polypeptide antibody. The polypeptide asantigen includes N-terminus peptides, C-terminus peptides or peptides ofcentral portions other than above-mentioned ligand polypeptides orpartial peptides thereof. To be more specifically includes the partialpeptide of SEQ ID NO: 92, 93 or 94.

The partial peptide may be a peptide containing each of the domains or apeptide containing a plurality of the domains within the molecule.

The partial peptide mentioned in this specification may be one endingwith an amide bond (—CONH₂) or an ester bond (—COOR) at the C-terminus.The ester here includes the same one of the above polypeptide. When thepartial peptide has a carboxyl or carboxylate group in any positionother than the C-terminus, the case in which such group or moiety hasbeen amidated or esterified also falls within the scope of the partialpeptide in the present invention. The ester here may be of the same oneas the above-mentioned ester at the C-terminus.

The ligand polypeptide or its partial peptide of the present inventionmay be in the form of a fused protein which fused with a protein whosefunctions or properties are already known.

The salt of such partial peptide of the ligand polypeptide of presentinvention may be of the same one as the above-mentioned salt of thepolypeptide.

The partial peptide of the ligand polypeptide of the invention, itsamide or ester, or a salt thereof can be produced by the same syntheticprocesses as mentioned for the polypeptide or by cleaving thepolypeptide of the present invention with a suitable peptidase.

The DNA coding for the ligand polypeptide or a partial peptide thereofof the present invention may be any DNA comprising the nucleotidesequence encoding a polypeptide having an amino acid sequence of SEQ IDNO:73 or its substantial equivalent thereto. Furthermore, the DNA may beany of genomic DNA, genomic DNA library, tissue- or cell-derived cDNA,tissue- or cell-derived cDNA library, and synthetic DNA. The vector forsuch as library may be any of bacteriophage, plasmide, cosmide, andphagimide. Moreover, it can be directly amplified by the RT-PCR methodby using an RNA fraction may be prepared from a tissue or cells.

To be more specific, as the DNA coding for a polypeptide derived fromrat whole brain or bovine hypothalmus and comprising the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:44, the DNA comprising thenucleotide sequence of SEQ ID NO:2 can be exemplified. In SEQ ID NO:2, Rat 129th position represents G or A, and Y at 179th and 240th positionsrepresents C or T. When Y at 179th position is C, the amino acidsequence of SEQ ID NO:1 is encoded, and when Y at 179th position is T,the amino acid sequence of SEQ ID NO:44 is encoded.

As the DNA coding for a bovine-derived polypeptide comprising the aminoacid sequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9 or 10, a DNA comprisingthe nucleotide sequence of SEQ ID NO:11, 12, 13, 14, 15, 16, 17 or 18can be exemplified. Here, R at 63th position of SEQ ID NO:11, 13, 14 or15 and R at 29th position of SEQ ID NO:12, 16, 17, or 18 represent G orA.

As the DNA coding for a rat-derived polypeptide of SEQ ID NO:45, 47, 48,49, 50, 51, or 52, a DNA comprising the nucleotide sequence of SEQ IDNO:46, 53, 54, 55, 56, 57, or 58 can be exemplified.

Furthermore, as the DNA coding for a human-derived peptide of SEQ IDNO:59, 61, 62, 63, 64, 65, or 66, a DNA comprising the nucleotidesequence of SEQ ID NO:60, 67, 68, 69, 70, 71, or 72 can be exemplified.

Among DNAs coding for the bovine-derived polypeptide comprising theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:44, the rat-derivedpolypeptide comprising the amino acid sequence of SEQ ID NO:45, or thehuman-derived polypeptide comprising the amino acid sequence of SEQ IDNO:59, DNA fragments comprising partial nucleotide sequences of 6 to 90,preferably 6 to 60, more preferably 9 to 30, and especially preferably12 to 30 can be advantageously used as DNA probes as well.

The DNA coding for the ligand polypeptide or a partial peptide thereofof the present invention can be produced by the following geneticengineering procedures.

The DNA fully encoding the polypeptide or partial peptide of the presentinvention can be cloned either by PCR amplification using synthetic DNAprimers having a partial nucleotide sequence of the polypeptide orpartial peptide or by hybridization using the DNA inserted in a suitablevector and labeled with a DNA fragment comprising a part or full regionof a human-derived polypeptide or a synthetic DNA. The hybridization canbe carried out typically by the procedure described in Molecular Cloning(2nd ed., J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Whena commercial library is used, the instructions given in the accompanyingmanual can be followed.

The cloned DNA coding for the polypeptide or partial peptide can be useddirectly or after digestion with a restriction enzyme or addition of alinker depending on purposes. This DNA has ATG as the translationinitiation codon at the 5′ end and may have TAA, TGA, or TAG as thetermination codon at the 3′ end. The translation initiation andtermination codons can be added by means of suitable DNA adapters.

An expression vector for the polypeptide or partial peptide can beproduced by, for example (a) cutting out a target DNA fragment from theDNA for the polypeptide or partial peptide of the present invention and(b) ligating the target DNA fragment with the downstream side of apromoter in a suitable expression vector.

The vector may include plasmids derived from Escherichia coli, e.g.,pBR322, pBR325, pUC12, pUC13, etc.; plasmids derived from Bacillussubtilis, e.g., pUB110, pTP5, pC194, etc.; plasmids derived from yeastse.g., pSH19, pSH15, etc.; bacteriophages such as λ-phage, and animalvirus such as retrovirus, vaccinia virus and baculovirus.

According to the present invention, any promoter can be used as long asit is compatible with the host cell which is used for expressing a gene.When the host for the transformation is E. coli, the promoters arepreferably trp promoters, lac promoters, recA promoters, λ_(PL)promoters, lpp promoters, etc. When the host for the transformation isBacillus, the promoters are preferably SPO1 promoters, SPO2 promoters,penP promoters, etc. When the host is a yeast, the promoters arepreferably PHO5 promoters, PGK promoters, GAP promoters, ADH promoters,etc. When the host is an animal cell, the promoters include SV40-derivedpromoters, retrovirus promoters, metallothionein promoters, heat shockpromoters, cytomegalovirus (CMV) promoters, SRα promoters, etc. Anenhancer can be effectively utilized for expression.

As required, furthermore, a host-compatible signal sequence is added tothe N-terminal side of the polypeptide or partial peptide thereof. Whenthe host is E. coli, the utilizable signal sequences may includealkaline phosphatase signal sequences, OmpA signal sequences, etc. Whenthe host is Bacillus, they may include α-amylase signal sequences,subtilisin signal sequences, etc. When the host is a yeast, they mayinclude mating factor α signal sequences, invertase signal sequences,etc. When the host is an animal cell, they may include insulin signalsequences, α-interferon signal sequences, antibody molecule signalsequences, etc.

A transformant or transfectant is produced by using the vector thusconstructed, which carries the polypeptide or partial peptide-encodingDNA of the present invention. The host may be, for example, Escherichiamicroorganisms, Bacillus microorganisms, yeasts, insect cells, animalcells, etc. Examples of the Escherichia and Bacillus microorganismsinclude Escherichia coli K12•DH1 [Proc. Natl. Acad. Sci. USA, Vol. 60,160 (1968)], JM103 [Nucleic Acids Research, Vol. 9, 309 (1981)], JA221[Journal of Molecular Biology, Vol. 120, 517 (1978)], HB101 [Journal ofmolecular Biology, Vol, 41, 459 (1969)], C600 [Genetics, Vol. 39, 440(1954)], etc.

Examples of the Bacillus microorganism are, for example Bacillussubtilis MI114 [Gene, Vol. 24, 255 (1983)], 207-21 [Journal ofBiochemistry, Vol. 95, 76 (1984)], etc.

The yeast may be, for example, Saccharomyces cerevisiae AH22, AH22R⁻,NA87-11A, DKD-5D, 20B-12, etc. The insect may include a silkworm (Bombyxmori larva), [Maeda et al, Nature, Vol. 315, 592 (1985)] etc. The hostanimal cell may be, for example, monkey-derived cell line, COS-7, Vero,Chinese hamster ovary cell line (CHO cell), DHFR gene-deficient Chinesehamster cell line (dhfr⁻ CHO cell), mouse L cell, mouse myeloma cell,human FL, etc.

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. Transformation of Escherichiamicroorganisms can be carried out in accordance with methods asdisclosed in, for example, Proc. Natl. Acad. Sci. USA, Vol. 69, 2110(1972), Gene, Vol. 17, 107 (1982), etc. Transformation of Bacillusmicroorganisms can be carried out in accordance with methods asdisclosed in, for example, Molecular & General Genetics, Vol. 168, 111(1979), etc. Transformation of the yeast can be carried out inaccordance with methods as disclosed in, for example, Proc. Natl. Acad.Sci. USA, Vol. 75, 1929 (1978), etc. The insect cells can be transformedin accordance with methods as disclosed in, for example, Bio/Technology,6, 47-55, 1988. The animal cells can be transformed by methods asdisclosed in, for example, Virology, Vol. 52, 456, 1973, etc. Thetransformants or transfectants wherein the expression vector carrying apolypeptide or partial peptide thereof encoding DNA harbors are producedaccording to the aforementioned techniques.

Cultivation of the transformant (transfectant) in which the host isEscherichia or Bacillus microorganism can be carried out suitably in aliquid culture medium. The culture medium may contains carbon sources,nitrogen sources, minerals, etc. necessary for growing the transformant.The carbon source may include glucose, dextrin, soluble starch, sucrose,etc. The nitrogen source may include organic or inorganic substancessuch as ammonium salts, nitrates, corn steep liquor, peptone, casein,meat extracts, bean-cakes, potato extracts, etc. Examples of theminerals may include calcium chloride, sodium dihydrogen phosphate,magnesium chloride, etc. It is further allowable to add yeasts,vitamines, growth-promoting factors, etc. It is desired that the culturemedium is pH from about 5 to about 8.

The Escherichia microorganism culture medium is preferably an M9 mediumcontaining, for example, glucose and casamino acid (Miller, Journal ofExperiments in Molecular Genetics), 431-433, Cold Spring HarborLaboratory, New York, 1972. Depending on necessity, the medium may besupplemented with drugs such as 3β-indolyl acrylic acid in order toimprove efficiency of the promoter. In the case of an Escherichia host,the cultivation is carried out usually at about 15 to 43° C. for about 3to 24 hours. As required, aeration and stirring may be applied. In thecase of Bacillus host, the cultivation is carried out usually at about30 to 40° C. for about 6 to 24 hours. As required, aeration and stirringmay be also applied. In the case of the transformant in which the hostis a yeast, the culture medium used may include, for example, aBurkholder minimum medium [Bostian, K. L. et al., Proc. Natl. Acad. Sci.USA, Vol. 77, 4505 (1980)], an SD medium containing 0.5% casamino acid[Bitter, G. A. et al., Proc. Natl. Acad. Sci. USA, Vol. 81, 5330(1984)], etc. It is preferable that the pH of the culture medium isadjusted to be from about 5 to about 8. The cultivation is carried outusually at about 20 to 35° C. for about 24 to 72 hours. As required,aeration and stirring may be applied. In the case of the transformant inwhich the host is an insect, the culture medium used may include thoseobtained by suitably adding additives such as passivated (orimmobilized) 10% bovine serum and the like to the Grace's insect medium(Grace, T. C. C., Nature, 195, 788 (1962)). It is preferable that the pHof the culture medium is adjusted to be about 6.2 to 6.4. Thecultivation is usually carried out at about 27° C. for about 3 to 5days. As desired, aeration and stirring may be applied. In the case ofthe transformant in which the host is an animal cell, the culture mediumused may include MEM medium [Science, Vol. 122, 501 (1952)], DMEM medium[Virology, Vol. 8, 396 (1959)], RPMI 1640 medium [Journal of theAmerican Medical Association, Vol. 199, 519 (1967)], 199 medium[Proceedings of the Society of the Biological Medicine, Vol. 73, 1(1950)], etc. which are containing, for example, about 5 to 20% of fetalcalf serum. It is preferable that the pH is from about 6 to about 8. Thecultivation is usually carried out at about 30 to 40° C. for about 15 to60 hours. As required, medium exchange, aeration and stirring may beapplied.

Separation and purification of the polypeptide or partial peptide fromthe above-mentioned cultures can be carried out according to methodsdescribed herein below.

To extract polypeptide or partial peptide from the culturedmicroorganisms or cells, the microorganisms or cells are collected byknown methods after the cultivation, suspended in a suitable buffersolution, disrupted by ultrasonic waves, lysozyme and/or freezing andthawing, etc. and, then, a crude extract of the polypeptide or partialpeptide is obtained by centrifugation or filtration. Other conventionalextracting or isolating methods can be applied. The buffer solution maycontain a protein-denaturing agent such as urea or guanidinehydrochloride or a surfactant such as Triton X-100 (registeredtrademark, hereinafter often referred to as “TM”).

In the case where the polypeptide or partial peptide are secreted intoculture media, supernatant liquids are separated from the microorganismsor cells after the cultivation is finished and the resulting supernatantliquid is collected by widely known methods. The culture supernatantliquid and extract containing the polypeptide or partial peptide can bepurified by suitable combinations of widely known methods forseparation, isolation and purification. The widely known methods ofseparation, isolation and purification may include methods whichutilizes solubility, such as salting out or sedimentation with solventsmethods which utilizes chiefly a difference in the molecular size orweight, such as dialysis, ultrafiltration, gel filtration andSDS-polyacrylamide gel electrophoresis, methods utilizing a differencein the electric charge, such as ion-exchange chromatography, methodsutilizing specific affinity such as affinity chromatography, methodsutilizing a difference in the hydrophobic property, such asreverse-phase high-performance liquid chromatography, and methodsutilizing a difference in the isoelectric point such as isoelectricelectrophoresis, or chromatofocusing, etc.

In cases where the polypeptide or partial peptide thus obtained is in afree form, the free protein can be converted into a salt thereof byknown methods or method analogous thereto. In case where the polypeptideor partial peptide thus obtained is in a salt form vice versa, theprotein salt can be converted into a free form or into any other saltthereof by known methods or method analogous thereto.

The polypeptide or partial peptide produced by the transformant can bearbitrarily modified or a polypeptide can be partly removed therefrom,by the action of a suitable protein-modifying enzyme before or after thepurification. The protein-modifying enzyme may include trypsin,chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase, etc.The activity of the polypeptide or partial peptide thus formed can bemeasured by experimenting the coupling (or binding) with receptor or byenzyme immunoassays (enzyme linked immunoassays) using specificantibodies.

The DNA coding for the ligand polypeptide of the present invention, theligand polypeptide or a partial peptide thereof can be used for (1)synthesis of a part or the full length of the ligand for Gprotein-coupled receptor protein, (2) search for the physiologicalactivities of the ligand polypeptide or partial peptide thereof of thepresent invention, (3) preparation of a synthetic oligonucleotide probeor a PCR primer, (4) acquisition of DNAs coding for ligands of Gprotein-coupled receptor proteins and precursor proteins, (5)development of receptor-binding assay systems using the expression ofrecombinant receptor proteins and screening of candidate medicinallyactive compounds, (6) acquisition of antibodies and antisera, (7)development of diagnostic agents utilizing said antibodies or antisera,(8) development of drugs such as pituitary function modulators, centralnervous system function modulators, and pancreatic function modulators,and (9) gene therapies, among other uses.

Particularly by using the receptor binding assay system using theexpression of a recombinant G protein-coupled receptor protein, which isdescribed hereinafter, agonists or antagonists of G protein-coupledreceptors which are specific to warm-blood animals including humans canbe screened and such agonists and antagonists can be used asprophylactic and therapeutic agents for various diseases.

Further, referring to (8) above, the ligand polypeptide, a partialpeptide thereof, or the DNA encording either of them of the presentinvention is useful as a safe pharmaceutical composition of low toxicpotential because it is recognized as a ligand by the G protein-coupledreceptor protein expressed in the hypophysis, central nervous system andpancreatic β cells. The ligand polypeptide, a partial peptide thereof,or the DNA encording either of them of the present invention isassociated with the modulation of pituitary function, central nervoussystem function, and pancreatic function and, therefore, can be used asa therapeutic and prophylactic pharmaceutical composition for dementiasuch as senile dementia, cerebrovascular dementia (dementia due tocerebrovascular disorder), dementia associated with phylodegenerativeretroplastic diseases (e.g. Alzheimer's disease, Parkinson's disease,Pick's disease, Huntington's disease, etc.), dementia due to infectiousdiseases (e.g. delayed viral infections such as Creutzfelt-Jakobdisease), dementia associated with endocrine, metabolic, and toxicdiseases (e.g. hypothyroidism, vitamin B12 deficiency, alcoholism, andpoisoning due to various drugs, metals, or organic compounds), dementiaassociated with oncogenous diseases (e.g. brain tumor), dementia due totraumatic diseases (e.g. chronic subdural hematoma):, depression(melancholia), hyperkinetic (microencephalo-pathy) syndrome, disturbanceof consciousness, anxiety syndrome, schizophrenia, horror, growthhormone secretory disease (e.g. gigantism, acromegalic gigantism etc.),hyperphagia, polyphagia, hypercholesterolemia, hyperglyceridemia,hyperlipemia, hyperprolactinemia, diabetes (e.g. diabetic complications,diabetic nephropathy, diabetic neurophathy, diabetic retinopathy etc.),cancer (e.g. mammary cancer, lymphatic leukemia, cystic cancer, ovarycancer, prostatic cancer etc.), pancreatitis, renal disease (e.g.chromic renal failure, nephritis etc.), Turner's syndrome, neurosis,rheumatoid arthritis, spinal injury, transient brain ischemia,amyotrophic lateral sclerosis, acute myocardial infarction,spinocerebellar degeneration, bone fracture, trauma, atopic dermatitis,osteoporosis, asthma, epilepsy, infertility or oligogalactia.Furthermore, they can be also used as the agent for improvement inpostoperative nutritional status and/or vasopressor.

When the polypeptide, a partial peptide thereof, or the DNA encodingeither of them of the present invention is used as a pharmaceuticalcomposition as described above, it can be used by conventional methods.For example, it can be used orally in the form of tablets which may besugar coated as necessary, capsules, elixirs, microcapsules etc., ornon-orally in the form of injectable preparations such as asepticsolutions and suspensions in water or other pharmaceutically acceptableliquids. These preparations can be produced by mixing the polypeptide, apartial peptide thereof, or the DNA encoding either of them withphysiologically acceptable carriers, flavoring agents, excipients,vehicles, antiseptics, stabilizers, binders etc. in unit dosage formsrequired for generally accepted manners of pharmaceutical making. Activeingredient contents in these preparations are set so that an appropriatedose within the specified range is obtained.

Additives which can be mixed in tablets, capsules etc. include binderssuch as gelation, corn starch, tragacanth and gum arabic, excipientssuch as crystalline cellulose, swelling agents such as corn starch,gelatin and alginic acid, lubricants such as magnesium stearate,sweetening agents such as sucrose, lactose and saccharin, and flavoringagents such as peppermint, akamono oil and cherry. When the unit dosageform is the capsule, the above-mentioned materials may furtherincorporate liquid carriers such as oils and fats. Sterile compositionsfor injection can be formulated by ordinary methods of pharmaceuticalmaking such as by dissolving or suspending active ingredients, naturallyoccuring vegetable oils such as sesame oil and coconut oil, etc. invehicles such as water for injection.

Aqueous liquids for injection include physiological saline and isotonicsolutions containing glucose and other auxiliary agents, e.g.,D-sorbitol, D-mannitol and sodium chloride, and may be used incombination with appropriate dissolution aids such as alcohols, e.g.,ethanol, polyalcohols, e.g., propylene glycol and polyethylene glycol,nonionic surfactants, e.g., polysorbate 80 (TM) and HCO-50 etc. Oilyliquids include sesame oil and soybean oil, and may be used incombination with dissolution aids such as benzyl benzoate and benzylalcohol. Furthermore the above-mentioned materials may also beformulated with buffers, e.g., phosphate buffer and sodium acetatebuffer; soothing agents, e.g., benzalkonium chloride, procainehydrochloride; stabilizers, e.g., human serum albumin, polyethyleneglycol; preservatives, e.g., benzyl alcohol, phenol; antioxidants etc.The thus-prepared injectable liquid is normally filled in an appropriateampule. Because the thus-obtained preparation is safe and of lowtoxicity, it can be administered to humans or warm-blooded mammals,e.g., mouse, rats, guinea pig, rabbits, chicken, sheep, pigs, bovines,cats, dogs, monkeys, baboons, chimpanzees, for instance.

The dose of said polypeptide, a partial peptide thereof, or the DNAencoding either of them is normally about 0.1-100 mg, preferably 1.0-50mg, and more preferably 1.0-20 mg per day for an adult (weighing 60 kg)in oral administration, depending on symptoms etc. In non-oraladministration, it is advantageous to administer the polypeptide, apartial peptide thereof, or the DNA encoding either of them in the formof injectable preparation at a daily dose of about 0.01-30 mg,preferably about 0.1-20 mg, and more preferably about 0.1-10 mg peradministration by an intravenous injection for an adult (weighing 60kg), depending on subject of administration, target organ, symptoms,method of administration etc. For other animal species, correspondingdoes as converted per 60 kg weight can be administered.

The G protein-coupled receptor protein for the above ligand polypeptideof the present invention may be any of G protein-coupled receptorproteins derived from various tissues, e.g. hypophysis, pancreas, brain,kidney, liver, gonad, thyroid gland, gall bladder, bone marrow, adrenalgland, skin, muscle, lung, alimentary canal, blood vessel, heart, etc.of human and other warm-blooded animals, e.g. guinea pig, rat, mouse,swine, sheep, bovine, monkey, etc.; and comprising an amino acidsequence of SEQ ID NO:19, 20, 21, 22 or 23, or substantial equivalentthereto. Thus, the G protein-coupled receptor protein of the presentinvention includes, in addition to proteins comprising the SEQ ID NO:19,20, 21, 22 or 23, those proteins comprising amino acid sequences ofabout 90-99.9% homology to the amino acid sequence of SEQ ID NO:19, 20,21, 22 or 23 and having qualitatively substantially equivalent activityto proteins comprising the amino acid sequence of SEQ ID NO:19, 20, 21,22, or 23. The activities which these proteins are possessed may includeligand binding activity and signal transduction activity. The term“substantially equivalent” means that the nature of the ligand bindingactivity and the like is equivalent. Therefore, it is allowable thateven differences among grades such as the strength of ligand bindingactivity and the molecular weight of receptor protein are present.

To be further specific, the G protein-coupled receptor proteins includehuman pituitary-derived G protein-coupled receptor proteins whichcomprises the amino acid sequence of SEQ ID NO:19 or/and SEQ ID NO:20,mouse pancreas-derived G protein-coupled receptor proteins whichcomprises the amino acid sequence of SEQ ID NO:22, and mousepancreas-derived G protein-coupled receptor proteins which comprises theamino acid sequence of SEQ ID NO:23. As the human pituitary-derived Gprotein-coupled receptor proteins which comprises the amino acidsequence of SEQ ID NO:19 and/or SEQ ID NO:20 include the humanpituitary-derived G protein-coupled receptor protein which comprises theamino acid sequence of SEQ ID NO:21. The G protein-coupled receptorproteins further include proteins wherein 1 to 30 amino acid residues,preferably 1 to 10 amino acid residues are deleted from the amino acidsequence of SEQ ID NO:19, 20, 21, 22 or 23, proteins wherein 1 to 30amino acid residues, preferably 1 to 10 amino acid residues are added tothe amino acid sequence of SEQ ID NO:19, 20, 21, 22, or 23, the proteinswherein 1 to 30 amino acid residues, preferably 1 to 10 amino acidresidues in the amino acid sequence of SEQ ID NO:19, 20, 21, 22, or 23are substituted with one or more other amino acid residues.

Here, the protein which comprises an amino acid sequence of SEQ ID NO:21or substantial equivalent thereto contains the full-length of the aminoacid sequence for human pituitary-derived G protein-coupled receptorprotein. The protein which comprises an amino acid sequence of SEQ IDNO:19 or/and SEQ ID NO:20 or substantial equivalent thereto may be apartial peptide of the protein which comprises an amino acid sequence ofSEQ ID NO:21 or substantial equivalent thereto. The protein whichcomprises an amino acid sequence of SEQ ID NO:22 or SEQ ID NO:23 orsubstantial equivalent thereto is a G protein-coupled receptor proteinwhich is derived from mouse pancreas but since its amino acid sequenceis quite similar to the amino acid sequence of SEQ ID NO:19 or/and SEQID NO:20 (cf. Example 8, FIG. 13 in particular), the protein whichcomprises an amino acid sequence of SEQ ID NO:22 or 23 or substantialequivalent thereto is also subsumed in the category of said partialpeptide of the protein which comprises an amino acid sequence of SEQ IDNO:21 or substantial equivalent thereto.

Thus, the above-mentioned protein comprising an amino acid sequence ofSEQ ID NO:21 or substantial equivalent thereto or a partial peptide ofthe protein or a salt thereof, which will be described below, includesthe protein comprising an amino acid sequence of SEQ ID NO:19, 20, 22,or 23 or substantial equivalent thereto, or a salt thereof.

Furthermore, the G protein-coupled receptor protein includes the proteinin which the N-terminal Met has been protected with a protective group,e.g. C₁₋₆ acyl such as formyl or acetyl, the protein in which theN-terminal side of Glu has been cleaved in vivo to form pyroglutamine,the protein in which the side chain of any relevant constituent aminoacid has been protected with a suitable protective group, e.g. C₁₋₆ acylsuch as formyl or acetyl, and the complex protein such as glycoproteinsavailable upon attachment of sugar chains.

The salt of G protein-coupled receptor protein includes the same kindsof salts as mentioned for the ligand polypeptide.

The G protein-coupled receptor protein or a salt thereof or a partialpeptide thereof can be produced from the tissues or cells of human orother warm-blooded animals by the per se known purification technologyor, as described above, by culturing a transformant carrying a DNAcoding for the G protein-coupled receptor protein. It can also beproduced in accordance with the procedures for peptide synthesis whichare described above.

A partial peptide of G protein-coupled receptor protein may include, forexample, a fragment containing an extracellular portion of the Gprotein-coupled receptor protein, i.e. the site which is exposed outsidethe cell membranes. Examples of the partial peptide are fragmentscontaining a region which is an extracellular area (hydrophilic region)as analyzed in a hydrophobic plotting analysis of the G protein-coupledreceptor protein, such as shown in FIG. 3, FIG. 4, FIG. 8, FIG. 11, orFIG. 14. Furthermore, a fragment which partly contains a hydrophobicregion may also be used. While peptides which separately contains eachdomain may be used too, peptides which contains multiple domains at thesame time will be used as well.

The salt of a partial peptide of G protein-coupled receptor protein maybe the same one of salt mentioned for the salt of ligand polypeptide.

The DNA coding for the G protein-coupled receptor protein may be any DNAcomprising a nucleotide sequence encoding the G protein-coupled receptorprotein which comprises an amino acid sequence of SEQ ID NO:19, 20, 21,22, or 23 or substantial equivalent thereto. It may also be any one ofgenomic DNA, genomic DNA library, tissue- or cell-derived cDNA, tissue-or cell-derived cDNA library, and synthetic DNA. The vector for such alibrary may include bacteriophage, plasmid, cosmid, and phargimide.Furthermore, using an RNA fraction prepared from a tissue or cells, adirect amplification can be carried out by the RT-PCR method.

To be specific, the DNA encoding the human pituitary-derived Gprotein-coupled receptor protein which comprises the amino acid sequenceof SEQ ID NO:19 include a DNA which comprises the nucleotide sequence ofSEQ ID NO:24. The DNA encoding the human pituitary-derived Gprotein-coupled receptor protein which comprises the amino acid sequenceof SEQ ID NO:20 include a DNA which comprises the nucleotide sequence ofSEQ ID NO:25. The DNA encoding the human pituitary-derived Gprotein-coupled receptor protein which comprises the amino acid sequenceof SEQ ID NO:21 include a DNA which comprises the nucleotide sequence ofSEQ ID NO:26. The DNA encoding the mouse pancreas-derived Gprotein-coupled receptor protein which comprises the amino acid sequenceof SEQ ID NO:22 include a DNA which comprises the nucleotide sequence ofSEQ ID NO:27. The DNA encoding the mouse pancreas-derived Gprotein-coupled receptor protein which comprises the amino acid sequenceof SEQ ID NO:23 include a DNA comprising the nucleotide sequence of SEQID NO:28.

A method for cloning the DNA completely coding for the G protein-coupledreceptor protein, vector, promoter, host cell, a method fortransformation, a method for culturing the transformant or a method forseparation and purification of the G protein-coupled receptor proteinmay include the same one as mentioned for the ligand polypeptide.

To be specific, the plasmid phGR3 obtained in Example 5, describedhereinafter, is digested with the restriction enzyme SalI and thetranslation frame for the full-length cDNA encoding hGR3 is isolated.This frame is subjected to ligation to, for example, the expressionvector pAKKO-111 for animal cell use which has been treated with BAP(bacterial alkaline phosphatase) after SalI digestion for inhibition ofautocyclization. After completion of the ligation reaction, a portion ofthe reaction mixture is used for transfection of, for example,Escherichia coli DH5. Among the transformants obtained, a transformantin which the cDNA coding for hGR3 has been inserted in the forwarddirection with respect to a promoter, such as SRα, which has beeninserted into the expression vector beforehand is selected by mappingafter cleavage with restriction enzymes or by nucleotide sequencing andthe plasmid DNA is prepared on a production scale.

The thus-constructed DNA of the expression vector is introduced into CHOdhfr⁻ cells using a kit for introducing a gene into animal cells by thecalcium phosphate method, the liposome method or the like to provide ahigh G protein-coupled receptor protein (hGR3) expression CHO cell line.

The resulting CHO cells are cultured in a nucleic acid-free screeningmedium in a CO₂ incubator at 37° C. using 5% CO₂ for 1-4 days so as togive the G protein-coupled receptor protein (hGR3).

The G protein-coupled receptor protein is purified from the above CHOcells using an affinity column prepared by conjugating an antibody tothe G protein-coupled receptor protein or a partial peptide thereof to asupport or an affinity column prepared by conjugating a ligand for the Gprotein-coupled receptor protein.

The activity of the G protein-coupled receptor protein thus formed canbe measured by experimenting the binding with a ligand or by enzymeimmunoassays using specific antibodies.

The G protein-coupled receptor protein, the partial peptide thereof andthe G protein-coupled receptor protein-encoding DNA can be used for:

-   1) determining a ligand to the G protein-coupled receptor protein,-   2) obtaining an antibody and an antiserum,-   3) constructing a system for expressing a recombinant receptor    protein,-   4) developing a receptor-binding assay system using the above    developing system and screening pharmaceutical candidate compounds,-   5) designing drugs based upon comparison with ligands and receptors    which have a similar or analogous structure,-   6) preparing a probe for the analysis of genes and preparing a PCR    primer,-   7) gene manipulation therapy,

In particular, it is possible to screen a G protein-coupled receptoragonist or antagonist specific to a warm-blooded animal such as humanbeing by a receptor-binding assay system which uses a system forexpressing a recombinant G protein-coupled receptor protein. The agonistor antagonist thus screened or characterized permits variousapplications including prevention and/or therapy of a variety ofdiseases.

Described below are uses of ligand polypeptide of the present invention,G protein-coupled receptor proteins to the ligand polypeptide, ligandpolypeptide-encoding DNAs, G protein-coupled receptor protein-encodingDNAs and their antibodies.

(1) Method for Determing a Ligand to the G Protein-Coupled receptorProtein

The G protein-coupled receptor protein, the partial peptide thereof or asalt thereof is useful as a reagent for investigating or determining aligand to said G protein-coupled receptor protein.

According to the present invention, methods for determining a ligand tothe G protein-coupled receptor protein which comprises contacting the Gprotein-coupled receptor protein or the partial peptide thereof with thecompound to be tested, and measuring the binding amount, the cellstimulating activity, etc. of the test compound to the G protein-coupledreceptor protein or the partial peptide thereof are provided.

The compound to be tested may include not only known ligands such asangiotensins, bombesins, canavinoids, cholecystokinins, glutamine,serotonin, melatonins, neuropeptides Y, opioids, purine, vasopressins,oxytocins, VIP (vasoactive intestinal and related peptides),somatostatins, dopamine, motilins, amylins, bradykinins, CGRP(calcitonin gene related peptides), leukotrienes, pancreastatins,prostaglandins, thromboxanes, adenosine, adrenaline, α- and β-chemokinessuch as IL-8, GROα, GROβ, GROγ, NAP-2, ENA-78, PF4, IP10, GCP-2, MCP-1,HC14, MCP-3, I-309, MIP1α, MIP-1β, RANTES, etc.; endothelins,enterogastrins, histamine, neurotensins, TRH, pancreatic polypeptides,galanin, modified derivatives thereof, analogues thereof, family membersthereof and the like but also tissue extracts, cell culturesupernatants, etc. of human or warm-blooded aminals such as mice, rats,swines, cattle, sheep and monkeys, etc. For example, said tissueextract, said cell culture supernatant, etc. is added to the Gprotein-coupled receptor protein for measurement of the cell stimulatingactivity, etc. and fractionated by relying on the measurements whereupona single ligand can be finally determined and obtained.

In one specific embodiment of the present invention, said method fordetermining the ligand includes a method for determining whether asample (including a compound or a salt thereof) is capable ofstimulating a target cell which comprises binding said compound with theG protein-coupled receptor protein either in the presence of the Gprotein-coupled receptor protein, the partial peptide thereof or a saltthereof, or in a receptor binding assay system in which the expressionsystem for the recombinant receptor protein is constructed and used; andmeasuring the receptor-mediated cell stimulating activity, etc. Examplesof said cell stimulating activities that can be measured includepromoting or inhibiting biological responses, e.g. liberation ofarachidonic acid, liberation of acetylcholine, liberation ofendocellular Ca²⁺, production of endocellular cAMP, production ofendocellular cGMP, production of inositol phosphate, changes in the cellmembrane potential, phosphorylation of endocellular protein, activationof c-fos, decrease in pH, etc, and preferably liberation of arachidonicacid. Examples of said compound or a salt thereof capable of stimulatingthe cell via binding with the G protein-coupled receptor protein includepeptides, proteins, nonpeptidic compounds, synthetic compounds,fermented products, etc.

In more specific embodiments of the present invention, said methods forscreening and identifying a ligand includes:

-   1) a method of screening for a ligand to a G protein-coupled    receptor protein, which comprises contacting a labeled test compound    with a G protein-coupled receptor protein or a salt thereof or its    partial peptide or a salt thereof, and measuring the amount of the    labeled test compound binding with said protein or salt thereof or    with said partial peptide or salt thereof;-   2) a method of screening for a ligand to a G protein-coupled    receptor protein, which comprises contacting a labeled test compound    with cells containing the G protein-coupled receptor protein or the    membrane fraction of said cell, and measuring the amount of the    labeled test compound binding with said cells or said membrane    fraction;-   3) a method of screening for a ligand to a G protein-coupled    receptor protein, which comprises contacting a labeled test compound    with the G protein-coupled receptor protein expressed on cell    membranes by culturing transformants carrying the G protein-coupled    receptor protein-encoding DNA and measuring the amount of the    labeled test compound binding with said G protein-coupled receptor    protein;-   4) a method of screening for a ligan to a G protein-coupled receptor    protein, which comprises contacting a test compound with cells    containing the G protein-coupled receptor protein, and measuring the    cell stimulating activity, e.g. promoting or inhibiting activity on    biological responses such as liberation of arachidonic acid,    liberation of acetylcholine, liberation of endocellular Ca²⁺,    production of endocullular cAMP, production of endocellular cGMP,    production of inositol phosphate, changes in the cell membrane    potential, phosphorylation of endocellular protein, activation of    c-fos, lowering in pH, etc. via the G protein-coupled receptor    protein; and-   5) a method of screening for a ligand to the G protein-coupled    receptor protein, which comprises contacting a test compound with    the G protein-coupled receptor protein expressed on the cell    membrane by culturing transformants carrying the G protein-coupled    receptor protein-encoding DNA, and measuring at least one cell    stimulating activity, e.g., an activity for promoting or inhibiting    physiological responses such as liberation of arachidonic acid,    liberation of 2+acetylcholine, liberation of endocellular Ca    production of endocellular cAMP, production of endocellular cGMP,    production of inositol phosphate, changes in the cell membrane    potential, phosphorylation of endocellular protein, activation of    c-fos, lowering in pH etc. via the G protein-coupled receptor    protein.

Described below are specific illustrations of the method for screeningand identifying ligands.

First, the G protein-coupled receptor protein used for the method fordetermining the ligand may include any material so far as it contains aG protein-coupled receptor protein, a partial peptide thereof or a saltthereof although it is preferable to express large amounts of the Gprotein-coupled receptor proteins in animal cells.

In the manufacture of the G protein-coupled receptor protein, theabove-mentioned method can be used and carried out by expressing saidprotein encoding DNA in mammalian cells or in insect cells. With respectto the DNA fragment coding for a particular region such as anextracellular epitope, the extracellular domains, etc., complementaryDNA may be used although the method of expression is not limitedthereto. For example, gene fragments or synthetic DNA may be used aswell.

In order to introduce the G protein-coupled receptor protein-encodingDNA fragment into host animal cells and to express it efficiently, it ispreferred that said DNA fragment is incorporated into the downstreamside of polyhedron promoters derived from nuclear polyhedrosis virusbelonging to baculovirus, promoters derived from SV40, promoters derivedfrom retrovirus, metallothionein promoters, human heat shock promoters,cytomegalovirus promoters, SRα promoters, etc. Examinations of thequantity and the quality of the expressed receptor can be carried out bymethods per se known to those of skill in the art or methods similarthereto based upon the present disclosure. For example, they may beconducted by methods described in publications such as Nambi, P. et al:The Journal of Biochemical Society, vol. 267, pages 19555-19559 (1992).

Accordingly, with respect to the determination of the ligand, thematerial containing a G protein-coupled receptor protein or partialpeptide thereof may include products containing G protein-coupledreceptor proteins which are purified by methods per se known to those ofskill in the art or methods similar thereto, peptide fragments of said Gprotein-coupled receptor protein, cells containing said Gprotein-coupled receptor protein, membrane fractions of the cellcontaining said protein, etc.

When the G protein-coupled receptor protein-containing cell is used inthe determining method of the ligand, said cell may be immobilized withbinding agents including glutaraldehyde, formalin, etc. Theimmobilization may be carried out by methods per se known to those ofskill in the art or methods similar thereto.

The G protein-coupled receptor protein-containing cells are host cellswhich express the G protein-coupled receptor protein. Examples of saidhost cells are microorganisms such as Escherichia coli, Bacillussubtilis, yeasts, insect cells, animal cells, etc.

The cell membrane fraction is a cell membrane-rich fraction which isprepared by methods per se known to those of skill in the art or methodssimilar thereto after disruption of cells. Examples of cell disruptionmay include a method for squeezing cells using a Potter-Elvehjemhomogenizer, a disruption by a Waring blender or a Polytron manufacturedby Kinematica, a disruption by ultrasonic waves, a disruption viablowing out cells from small nozzles together with applying a pressureusing a French press or the like, etc. In the fractionation of the cellmembrane, a fractionation method by means of centrifugal force such as afractional centrifugal separation and a density gradient centrifugalseparation is mainly used. For example, disrupted cellular liquid iscentrifuged at a low speed (500 rpm to 3,000 rom) for a short period(usually, from about one to ten minutes), the supernatant liquid isfurther centrifuged at a high speed (15,000 rpm to 30,000 rom) usuallyfor 30 minutes to two hours and the resulting precipitate is used as amembrane fraction. Said membrane fraction contains a lot of theexpressed G protein-coupled receptor protein and a lot of membranecomponents such as phospholipids and membrane proteins derived from thecells.

The amount of the G protein-coupled receptor protein in the membranefraction cell containing said G protein-coupled receptor protein ispreferably 10³ to 10⁸ molecules per cell or, more preferably, 10⁵ to 10⁷molecules per cell. Incidentally, the greater the expressed amount, thehigher the ligand binding activity (specific activity) per membranefraction whereby the construction of a highly sensitive screening systembecomes possible and, moreover, it permits measurement of a large amountof samples within the same lot.

In conducting the above-mentioned methods 1) to 3) wherein ligandscapable of binding with the G protein-coupled receptor protein aredetermined, a suitable G protein-coupled receptor fraction and a labeledtest compound are necessary. The G protein-coupled receptor fraction ispreferably a naturally occurring (natural type) G protein-coupledreceptor, a recombinant G protein-coupled receptor having the activityequivalent to that of the natural type. Here, the term “activityequivalent to” means the equivalent ligand binding activity, etc. asdiscussed above.

Suitable examples of the labeled test compound include above-mentionedcompound to be tested which are labeled with [³H], [¹²⁵I], [¹⁴C], [³⁵S],etc.

Specifically, the determination of ligands capable of binding with Gprotein-coupled receptor proteins is carried out as follows:

First, cells or cell membrane fractions containing the G protein-coupledreceptor protein are suspended in a buffer suitable for the assay toprepare the receptor sample for conducting the method of determining theligand binding with the G protein-coupled receptor protein. The buffermay include any buffer such as Tris-HCL buffer or phosphate buffer withpH 4-10, preferably, pH 6-8, etc., as long as it does not inhibit thebinding of the ligand with the receptor. In addition, surface-activeagents such as CHAPS, Tween 80™ (Kao-Atlas, Japan), digitonin,deoxycholate, etc. and various proteins such as bovine serum albumin(BSA), gelatin, milk derivatives, etc. may be added to the buffer withan object of descreasing the non-specific binding. Further, a proteaseinhibitor such as PMSF, leupeptin, E-64 (manufactured by PeptideLaboratory), pepstatin, etc. may be added with an object of inhibitingthe decomposition of the receptor and the ligand by protease. A testcompound labeled with a predetermined (or certain) amount (5,000 cpm to500,000 cpm) of [³H], [¹²⁵I]. [¹⁴C], [³⁵S], etc. coexists in 0.01 ml to10 ml of said receptor solution. In order to know the non-specificbinding amount (NSB), a reaction tube to which a great excessive amountof the unlabeled test compound is added is prepared as well. Thereaction is carried out at 0-50° C., preferably at 4-37° C. for 20minutes to 24 hours, preferably 30 minutes to three hours. After thereaction, it is filtered through a glass fiber filter or the like,washed with a suitable amount of the same buffer and the radioactivityremaining in the glass fiber filter is measured by means of a liquidscintillation counter or a gamma-counter. The test compound in which thecound (B-NSB) obtained by subtracting the non-specific binding amount(NSB) from the total binding amount (B) is more than 0 cpm is identifiedas a ligand to the G protein-coupled receptor protein.

In conducting the above-mentioned methods 4) to 5) wherein ligandscapable of binding with the G protein-coupled receptor protein aredetermined, the cell stimulating activity, e.g. the liberation ofarachidonic acid, the liberation of acetylcholine, endocellular Ca²⁺liberation, endocellular cAMP production, the production of inositolphosphate, changes in the cell membrane potential, the phosphorylationof endocellular protein, the activation of c-fos, lowering of pH, theactivation of G protein, cell promulgation, etc.; mediated by the Gprotein-coupled receptor protein may be measured by known methods or bythe use of commercially available measuring kits. To be more specific, Gprotein-coupled receptor protein-containing cells are at first culturedin a multi-well plate or the like.

In conducting the determination of ligand, it is substituted with afresh medium or a suitable buffer which does not show toxicity to thecells in advance of the experiment, and incubated under appropriateconditions and for sufficient time after adding a test compound, etc.thereto. Then, the cells are extracted or the supernatant liquid isrecovered and the resulting product is determined by each of themethods. When it is difficult to identify the production of thesubstance, e.g. arachidonic acid, etc. which is to be an index for thecell stimulating activity due to the decomposing enzyme contained in thecell, an assay may be carried out by adding an inhibitor against saiddecomposing enzyme. With respect to an activity such as an inhibitoryaction against cAMP production, it may be detected as an inhibitoryaction against the production of the cells whose fundamental productionis increased by forskolin or the like.

The kit used for the method of determining the ligand binding with the Gprotein-coupled receptor protein includes a G protein-coupled receptorprotein or a partial peptide thereof, cells containing the Gprotein-coupled receptor protein, a membrane fraction from the cellscontaining the G protein-coupled receptor protein, etc.

Examples of the kit for determining the ligand are as follows:

1. Reagent for Determing the Ligand.

1) Buffer for Measurement and Buffer for Washing.

The buffering product wherein 0.05% of bovine serum albumin(manufactured by Sigma) is added to Hanks' Balanced Salt Solution(manufactured by Gibco).

This product may be sterilized by filtration through a membrane filterwith a 0.45 μm pore size, and stored at 4° C. or may be formulated uponuse.

2) G Protein-Coupled Receptor Protein Sample.

CHO cells in which G protein-coupled receptor proteins are expressed aresubcultured at the rate of 5×10⁵ cells/well in a 12-well plate andcultured at 37° C. in a humidified 5% CO₂/95% air atmosphere for twodays to prepare the sample.

3) Labeled Test Compound.

The compound which is labeled with commercially available [³H], [¹²⁵I],[¹⁴C], [³⁵S], etc. or labeled with a suitable method.

The product in a state of an aqueous solution is stored at 4° C. or at−20° C. and, upon use, diluted to 1 μM with a buffer for themeasurement. In the case of a test compound which is barely soluble inwater, it may be dissolved in an organic solvent such asdimethylformamide, DMSO, methanol and the like.

4) Unlabeled Test Compound.

The same compound as the labeled one is prepared in a concentration of100 to 1,000-fold concentrated state.

2. Method of Measurement

-   1) G protein-coupled receptor protein-expressing CHO cells cultured    in a 12-well tissue culture plate are washed twice with 1 ml of    buffer for the measurement and then 490 μl of buffer for the    measurement is added to each well.-   2) Five μl of the labeled test compound is added and the mixture is    made to react at room temperature for one hour. For measuring the    nonspecific binding amount, 5 μl of the unlabeled test compound is    added.-   3) The reaction solution is removed from each well, which is washed    with 1 ml of a buffer for the measurement three times. The labeled    test compound which is binding with the cells is dissolved in 0.2N    NaOH-1% SDS and mixed with 4 ml of a liquid scintillator A    manufactured by WAKO Pure Chemical, Japan.-   4) Radioactivity is measured using a liquid scintillation counter    such as one manufactured by Beckmann.    (2) Prophylactic and Therapeutic Agent for G Protein-Coupled    Receptor Protein or Ligand Polypeptide Deficiency Diseases

If a ligand to the G protein-coupled receptor protein is revealed viathe aforementioned method (1), the ligand or the G protein-coupledreceptor protein-encoding DNA can be used as a prophylactic and/ortherapeutic agent for treating said G protein-coupled receptor proteinor ligand polypeptide deficiency diseases depending upon the action thatsaid ligand exerts.

For example, when there is a patient for whom the physiological actionof the ligand, e.g. pituitary function modulating action, centralnervious system function modulating action or pancreatic functionmodulating action; cannot be expected because of a descrease in the Gprotein-coupled receptor protein or ligand polypeptide in vivo, theamount of the G protein-coupled receptor protein or ligand polypeptidein the brain cells of said patient can be increased whereby the actionof the ligand can be fully achieved by:

-   (a) administering the G protein-coupled receptor protein-encoding    DNA to the patient to express it; or-   (b) inserting the G protein-coupled receptor protein or ligand    polypeptide-encoding DNA into brain cells or the like to said    patient. Accordingly, the G protein-coupled receptor protein- or    ligand polypeptide-encoding DNA can be used as a safe and less toxic    preventive and therapeutic agent for the G protein-coupled receptor    protein or ligand polypeptide deficiency diseases.

When the above-mentioned DNA is used as the above-mentioned agent, saidDNA may be used alone or after inserting it into a suitable vector suchas retrovirus vector, adenovirus vector, adenovirus-associated virusvector, etc. followed by subjecting the product vector to a conventionalmeans which is the same means as using the DNA coding for the ligandpolypeptide or partial peptide thereof as the pharmaceuticalcomposition.

(3) Quantitative Determination of the G Protein-Coupled Receptor Proteinto the Ligand Polypeptide

The ligand polypeptide that has a binding property for a Gprotein-coupled receptor protein or a partial peptide thereof, or a saltthereof are capable of determining quantitatively an amount of a Gprotein-coupled receptor protein or a partial peptide thereof, or a saltthereof in vivo with good sensitivity.

This quantitative determination may be carried out by, for example,combining with a competitive analysis. Thus, a sample to be determinedis contacted with the ligand polypeptide so that the concentration of aG protein-coupled receptor protein or a partial peptide thereof in saidsample can be determined. In one embodiment of the quantitativedetermination, the protocols described in the following 1) and 2) ormethods similar thereto may be used:

-   1) Hiroshi Irie (ed): “Radioimmunoassay” (Kodansha, Japan, 1974);    and-   2) Hiroshi Irie (ed): “Radioimmunoassay, Second Series” (Kodansha,    Japan, 1979).    (4) Screening of Compound Changing the Binding Activity of Ligand    Polypeptide, Partial Peptide Thereof or Salt Thereof (Hereinafter    Sometimes Referred to Briefly as Ligand or Ligand Polypeptide) with    the G Protein-Coupled Receptor Protein

G protein-coupled receptor proteins or partial peptide or salt thereofcan be used. Alternatively, expression systems for recombinant Gprotein-coupled receptor proteins are constructed and receptor bindingassay systems using said expression system are used. In these assaysystems, it is possible to screen compounds, e.g. peptides, proteins,nonpeptidic compounds, synthetic compounds, formented products, cellextracts, animal tissue extracts, etc.; or salts thereof which changesthe binding activity of a ligand polypeptide with the G protein-coupledreceptor protein. Such a compound includes a compound exhibiting a Gprotein-coupled receptor-mediated cell stimulating activity, e.g.activity of promoting or activity of inhibiting physiological reactionsincluding liberation of arachidonic acid, liberation of acetylchloline,endocellular Ca²⁺ liberation, endocellular cAMP production, endocellularcGMP production, production of inositol phosphate, changes in cellmembrane potential, phosphorylation of endocellular protein's activationof c-fos, lowering of pH, activation of G protein, cell promulgation,etc.; so-called “G protein-coupled receptor-agonist”, a compound freefrom such a cell stimulating activity, so-called “G protein coupledreceotor-antagonist”, etc. The term of “change the binding activity of aligand polypeptide” includes the both concept of the case in which thebinding of ligand is inhibited and the case in which the binding ofligand is promoted.

Thus, the present invention provides a method of screening for acompound which changes the binding activity of a ligand with a Gprotein-coupled receptor protein or a salt thereof, characterized bycomparing the following two cases:

-   (i) the case wherein the ligand is contacted with the G    protein-coupled receptor protein or salt thereof, or a partial    peptide thereof or a salt thereof; and-   (ii) the case wherein the ligand is contacted with a mixture of the    G protein-coupled receptor protein or salt thereof or the partial    peptide or salt thereof and said test compound.

In said screening method, one characteristic feature of the presentinvention resides in that the amount of the ligand bonded with said Gprotein-coupled receptor protein or the partial peptide thereof, thecell stimulating activity of the ligand, etc. are measured in both thecase where (i) the ligand polypeptide is contacted with Gprotein-coupled receptor proteins or partial peptide thereof and in thecase where (ii) the ligand polypeptide and the test compound arecontacted with the G protein-coupled receptor protein or the partialpeptide thereof, respectively and then compared therebetween.

In one more specific embodiment of the present invention, the followingis provided:

-   1) a method of screening for a compound or a salt thereof which    changes the binding activity of a ligand polypeptide with a G    protein-coupled receptor protein, characterized in that, when a    labeled ligand polypeptide is contacted with a G protein-coupled    receptor protein or a partial peptide thereof and when a labeled    ligand polypeptide and a test compound are contacted with a G    protein-coupled receptor protein or a partial peptide thereof, the    amounts of the labeled ligand polypeptide bonded with said protein    or a partial peptide thereof or a salt thereof are measured and    compared;-   2) a method of screening for a compound or a salt thereof which    changes the binding activity of a ligand polypeptide with a G    protein-coupled receptor protein, characterized in that, when a    labeled ligand polypeptide is contacted with cells containing G    protein-coupled receptor proteins or a membrane fraction of said    cells and when a labeled ligand polypeptide and a test compound are    contacted with cells containing G protein-coupled receptor proteins    or a membrane fraction of said cells, the amounts of the labeled    ligand polypeptide binding with said protein or a partial peptide    thereof or a salt thereof are measued and compared;-   3) a method of screening for a compound or a salt thereof which    changes the binding activity of a ligand polypeptide with a G    protein-coupled receptor protein, characterized in that, when a    labeled ligand polypeptide is contacted with G protein-coupled    receptor proteins expressed on the cell memberane by culturing a    transformant carrying a G protein-coupled receptor protein-encoding    DNA and when a labeled ligand polypeptide and a test compound are    contacted with G protein-coupled receptor proteins expressed on the    cell membrane by culturing a transformant carrying a G    protein-coupled receptor protein-encoding DNA, the amounts of the    labeled ligand polypeptide binding with said G protein-coupled    receptor protein are measured and compared;-   4) a method of screening for a compound or a salt thereof which    changes the binding of a ligand polypeptide with a G protein-coupled    receptor protein, characterized in that, when a G protein-coupled    receptor protein-activating compound, e.g. a ligand polypeptide of    the present invention, etc. is contacted with cells containing G    protein-coupled receptor proteins and when the G protein-coupled    receptor protein-activating compound and a test compound are    contacted with cells containing G protein-coupled receptor proteins,    the resulting G protein-coupled receptor protein-mediated cell    stimulting activities, e.g. activities of promoting or activities of    inhibiting physiological responses including liberation of    arachidonic acid, liberation of acetylcholine, endocellular Ca²⁺    liberation, endocellular cAMP production, endocellular cGMP    production, production of inositol phosphate, changes in cell    membrane potential, phosphorylation of endocellular proteins,    activation of c-fos, lowering of pH, activation of G protein, cell    promulgation, etc.; are measured and compared; and-   5) a method of screening for a compound or a salt thereof which    changes the binding activity of a ligand polypeptide with a G    protein-coupled receptor protein, characterized in that, when a G    protein-coupled receptor protein-activating compound, e.g. a ligand    polypeptide of the present invention, etc. is contacted with G    protein-coupled receptor proteins expressed on cell membranes by    culturing transformants carrying G protein-coupled receptor    protein-encoding DNA and when a G protein-coupled receptor    protein-activating compound and a test compound are contacted with    the G protein-coupled receptor protein expressed on the cell    membrane by culturing the transformant carrying the G    protein-coupled receptor protein-encoding DNA, the resulting G    protein-coupled receptor protein-mediated cell stimulating    activities, e.g. activities of promoting or activities of inhibiting    physiological responses such as liberation of arachidonic acid,    liberation of acetylcholine, endocellular Ca²⁺ liberation,    endocellular cAMP production, endocellular cGMP production,    production of inositol phosphate, changes in cell membrane    potential, phosphorylation of endocellular proteins, activation of    c-fos, lowering of pH, activation of G protein, and cell    promulgation, etc.; are measured and compared.

The G protein-coupled receptor agonist or antagonist have to be screenedby, first, obtaining a candidate compound by using G protein-coupledreceptor protein-containing cells, tissues or cell membrane fractionsderived from rat or the like (primary screening), then, making surewhether the candidate compound really inhibitis the binding betweenhuman G protein-coupled receptor proteins and ligands (secondaryscreening). Other receptor proteins inevitably exist and when the cells,the tissues or the cell membrane fractions were used, they intrinsicallymake it difficult to screen agonists or antagonists to the desiredreceptor proteins. By using the human-derived G protein-coupled receptorprotein, however, there is no need of effecting the primary screening,whereby it is possible to efficiently screen a compound that changes thebinding activity between a ligand and a G protein-coupled receptorAdditionally, it is possible to evaluate whether the compound that isscreened is a G protein-coupled receptor agonist or a G protein-coupledreceptor antagonist.

Specific explanations of the screening method will be given ashereunder.

First, with respect to the G protein-coupled receptor protein used forthe screening method of the present invention, any product may be usedso far as it contains G protein-coupled receptor proteins or partialpeptides thereof although the use of a membrane fraction of mammalianorgans is preferable. However, human organs can be extremely scarce and,accordingly, G protein-coupled receptor proteins which are expressed ina large amount using a recombinant technique are suitable for thescreening.

In the manufacture of the G protein-coupled receptor protein, theabove-mentioned method can be used.

When the G protein-coupled receptor protein-containing cells or cellmembrane fractions are used in the screening method, the above-mentionedmethod can be used.

In conducting the above-mentioned methods 1) to 3) for screening thecompound capable of changing the binding activity of the ligand with theG protein-coupled receptor protein, a suitable G protein-coupledreceptor fraction and a labeled ligand polypeptide are necessary. Withrespect to the G protein-coupled receptor fraction, it is preferred touse naturally occurring G protein-coupled receptors (natural type Gprotein-coupled receptors) or recombinant type G protein-coupledreceptor fractions with the activity equivalent to that of the naturaltype G protein coupled. Here the term “activity equivalent to” means thesame ligand binding activity, or the substantially equivalent ligandbinding activity.

With respect to the labeled ligand, it is possible to use labeledligands, labeled ligand amalogized compounds, etc. For example, ligandslabeled with [³H], [¹²⁵I], [¹⁴C], [³⁵S], etc. and other labeledsubstances may be utilized.

Specifically, G protein-coupled receptor protein-containing cells orcell membrane fractions are first suspended in a buffer which issuitable for the determining method to prepare the receptor sample inconducting the screening for a compound which changes the bindingactivity of the ligand with the G protein-coupled receptor protein. Withrespect to the buffer, any buffer such as Tris-HCl buffer or phosphatebuffer of pH 4-10, preferably, pH 6-8 which does not inhibit the bindingof the ligand with the receptor may be used.

In addition, a surface-active agent such as CHAPS, Tween 80™ (Kao-Atlas,Japan), digitonin, deoxycholate, etc. and/or various proteins such asbovine serum albumin (BSA), gelatine, etc. may be added to the bufferwith an object of decreasing the nonspecific binding. Further, aprotease inhibitor such as PMSF, leupeptin, E-64 manufactured by PeptideLaboratory, Japan, pepstatin, etc. may be added with an object ofinhibiting the decomposition of the receptor and the ligand by protease.A labeled ligand in a certain amount (5,000 cpm to 500,000 cpm) is addedto 0.01 ml to 10 ml of said receptor solution and, at the same time,10⁻⁴M to 10⁻¹⁰M of a test compound coexists. In order to determine thenonspecific binding amount (NSB), a reaction tube to which a greatexcessive amount of unlabeled test compounds is added is prepared aswell.

The reaction is carried out at 0-50° C., preferably at 4-37° C. for 20minutes to 24 hours, preferably 30 minutes to three hours. After thereaction, it is filtered through a glass fiber filter, a filter paper,or the like, washed with a suitable amount of the same buffer and theradioactivity retained in the glass fiber filter, etc. is measured bymeans of a liquid scintillation counter of a gamma-counter. Supposingthat the count (B₀-NSB) obtained by subtracting the nonspecific bindingamount (NSB) from the total binding amount (B₀) wherein an antagonizingsubstance is not present is set at 100%, a test compound in which thespecific binding amount (B-NSB) obtained by subtracting the nonspecificbinding amount (NSB) from the total binding amount (B) is, for example,less than 50% may be selected as a candidate ligand to the Gprotein-coupled receptor protein of the present invention.

In conducting the above-mentioned methods 4) to 5) for screening thecompound which changes the binding activity of the ligand with the Gprotein-coupled receptor protein, the G protein-coupled receptorprotein-mediated cell stimulating activity, e.g. activities of promotingor activities of inhibiting physiological responses such as release ofarachidonic acid, release of acetylcholine, intracellular Ca²⁺ increase,intracellular cAMP production, production of inositol phosphate, changesin the cell membrane potential, phosphorylation of intracullularproteins, activation of c-fos, lowering of pH, activation of G proteinand cell proliferation, etc.; may be measured by known methods or by theuse of commercially available measuring kits. To be more specific, Gprotein-coupled receptor protein-containing cells are at first culturedin a multiwell plate or the like.

In conducting the screening, it is substituted with a suitable bufferwhich does not show toxicity to fresh media or cells in advance,incubated under appropriate conditions and for a specified time afteradditing a test compound, etc. thereto. The resultant cells areextracted or the supernatant liquid is recovered and the resultingproduct is determined, preferably quantitatively, by each of themethods. When it is difficult to identify the production of theindicative substance, e.g. arachidonic acid, etc. which is to be anindication for the cell stimulating activity due to the presence ofdecomposing enzymes contained in the cell, an assay may be carried outby adding an inhibitor against said decomposing enzyme. With respect tothe activities such as an inhibitory action against cAMP production, itmay be detected as an inhibitory action against the cAMP production inthe cells whose fundamental production has been increased by forskolinor the like.

In conducting a screening by measuring the cell stimulating activity,cells in which a suitable G protein-coupled receptor protein isexpressed are necessary. Preferred G protein-coupled receptorprotein-expressing cells are naturally occurring G protein-coupledreceptor protein (natural type G protein-coupled receptorprotein)-containing cell lines or strains, e.g. mouse pancreatic β cellline, MIN6, etc., the above-mentioned recombinant type G protein-coupledreceptor protein-expressing cell lines or strains, etc.

Examples of the test compound includes peptide, proteins, non-peptidiccompounds, synthesized compounds, fermented products, cell extracts,plant extracts, animal tissue extracts, serum, blood, body fluid, etc.Those compounds may be novel or known.

A kit for screening the compound which changes the binding activity ofthe ligand with the G protein-coupled receptor protein or a salt thereofcomprises a G protein-coupled receptor protein or a partial peptidethereof, or G protein-coupled receptor protein-containing cells or cellmembrane fraction thereof.

Examples of the screening kit include as follows:

1. Reagent for Determining Ligand.

1) Buffer for Measurement and Buffer for Washing.

The product wherein 0.05% of bovine serum albumin (manufactured bySigma) is added to Hanks' Balanced Salt Solution (manufactured byGibco).

This may be sterilized by filtration through a membrane filter with a0.45 μm pore size, and stored at 4° C. or may be prepared upon use.

2) Sample of G Protein-Coupled Receptor Protein.

CHO cells in which a G protein-coupled receptor protein is expressed aresubcultured at the rate of 5×10⁵ cells/well in a 12-well plate andcultured at 37° C. with a 5% CO₂ and 95% air atmosphere for two days toprepare the sample.

3) Labeled Ligand.

The ligand which is labeled with commercially available [³H], [¹²⁵I],[¹⁴C], [³⁵S], etc.

The product in a state of an aqueous solution is stored at 4° C. or at−20° C. and, upon use, diluted to 1 μM with a buffer for themeasurement.

4) Standard Ligand Solution.

Ligand is dissolved in PBS containing 0.1% of bovine serum albumin(manufactured by Sigma) to make 1 mM and stored at −20° C.

2. Method of the Measurement.

-   1) CHO cells are cultured in a 12-well tissue culture plate to    express G protein-coupled receptor proteins. The G protein-coupled    receptor protein-expressing CHO cells are washed with 1 ml of buffer    for the measurement twice. Then 490 μl of buffer for the measurement    is added to each well.-   2) Five μl of a test compound solution of 10⁻³ to 10⁻¹⁰ M is added,    then 5 μl of a labeled ligand is added and is made to react at room    temperature for one hour. For knowing the non-specific binding    amount, 5 μl of the ligand of 10⁻³ M is added instead of the test    compound.

3) The reaction solution is removed from the well, which is washed with1 ml of buffer for the measurement three times. The labeled ligandbinding with the cells is dissolved in 0.2N NaOH-1% SDS and mixed with 4ml of a liquid scintillator A (such as manufactured by Wako PureChemical, Japan).

-   4) Radioactivity is measured using a liquid scintillation counter    (e.g., one manufactured by Beckmann) and PMB (percent maximum    binding) is calculated by the following equation:    PMB=[(B−NSB)/(B ₀ −NSB)]×100-   PMB: Percent maximum binding-   B: Value when a sample is added-   NSB: Nonspecific binding-   B₀: Maximum binding

The compound or a salt thereof obtained by the screening method or bythe screening kit is a compound which changes the binding activity of aligand polypeptide with a G protein-coupled receptor protein, whereinthe compound inhibits or promotes the bonding, and, more particularly,it is a compound having a cell stimulating activity mediated via a Gprotein-coupled receptor or a salt thereof, so-called “G protein-coupledreceptor agonist” or a compound having no said stimulating activity,so-called “G protein-coupled receptor antagonist”. Examples of saidcompound are peptides, proteins, non-peptidic compounds, synthesizedcompounds, fermented products, etc. and the compound may be novel orknown.

Said G protein coupled eceptor agonist has the same physiological actionas the ligand to the G protein-coupled receptor protein has and,therefore, it is useful as a safe and less toxic pharmaceuticalcomposition depending upon said ligand activity.

On the other hand, said G protein-coupled receptor antagonist is capableof inhibiting the physiological activity of the ligand to the Gprotein-coupled receptor protein and, therefore, it is useful as a safeand less toxic pharmaceutical composition for inhibiting said ligandactivity.

The ligand polypeptide of the present invention relates to the pituitaryfunction modulating action, central nervous system function modulatingaction or pancreatic function modulating action. Therefore, theabove-mentioned agonist or antagonist can be used as a therapeuticand/or prophylactic agent for dementia such as senile dementia,cerebrovascular dementia (dementia due to cerebrovascular disorder),dementia associated with phylodegenerative retroplastic diseases (e.g.Alzheimer's disease, Parkinson's disease, Pick's disease, Huntington'sdisease, etc.), dementia due to infectious diseases (e.g. delayed viralinfections such as Creutzfelt-Jakob disease), dementia associated withendocrine, metabolic, and toxic diseases (e.g. hypothyroidism, vitaminB12 deficiency, alcoholism, and poisoning due to various drugs, metals,or organic compounds), dementia associated with oncogenous diseases(e.g. brain tumor), dementia due to traumatic diseases (e.g. chronicsubdural hematoma):, depression (melancholia), hyperkinetic(microencephalo-pathy) syndrome, disturbance of consciousness, anxietysyndrome, schizophrenia, horror, growth hormone secretory disease (e.g.gigantism, acromegalic gigantism etc.), hyperphagia, polyphagia,hypercholesterolemia, hyperglyceridemia, hyperlipemia,hyperprolactinemia, hypoglycemia, pituitarism, pituitary drawfism,diabetes (e.g. diabetic complications, diabetic nephropathy, diabeticneurophathy, diabetic retinopathy etc.), cancer (e.g. mammary cancer,lymphatic leukemia, cystic cancer, ovary cancer, prostatic cancer etc.),pancreatitis, renal disease (e.g. chromic renal failure, nephritisetc.), Turner's syndrome, neurosis, rheumatoid arthritis, spinal injury,transient brain ischemia, amyotrophic lateral sclerosis, acutemyocardial infarction, spinocerebellar degeneration, bone fracture,trauma, atopic dermatitis, osteoporosis, asthma, epilepsy, infertilityor oligogalactia. Furthermore, the agonist or antagonist can be alsoused as hypnotic-sedative, agent for improvement in postoperativenutritional status, vasopressor or depressor.

When the compound or the salt thereof obtained by the screening methodor by the screening kit is used as the pharmaceutical composition, aconventional means which is the same means as using above-mentionedligand polypeptide as the pharmaceutical compoisiton may be appliedtherefor.

(5) Manufacture of Antibody or Antiserum Against the Ligand Polypeptideor the G Protein-Coupled Receptor Protein.

Antibodies, e.g. polyclonal antibody, monoclonal antibody, and antiseraagainst the ligand polypeptide or the G protein-coupled receptor proteinmay be manufactured by antibody- or antiserum-manufacturing methods perse known to those of skill in the art or methods similar thereto, usingthe ligand polypeptide or the G protein-coupled receptor protein asantigen. For example, polyclonal antibodies can be manufactured by themethod as given below.

[Preparation of a Polyclonal Antibody]

The above-mentioned polypeptide or protein as the antigen is coupled toa carrier protein. The carrier protein may for example be bovinethyroglobulin, bovine serum albumin, bovine gamma-globulin, hemocyanine,or Freund's complete adjuvant (Difco).

The coupling reaction between the antigen protein and the carrierprotein can be carried out by the known procedure. The reagent for usein the coupling reaction includes but is not limited to glutaraldehydeand water-soluble carbodiimide. The suitable ratio of the antigenprotein to the carrier protein is about 1:1 through about 1:10 and as tothe reaction pH, satisfactory results are obtained in many cases whenthe reaction is carried out around neutral, particularly in the range ofpH about 6-8. The reaction time is preferably about 1 to 12 hours inmany cases and more preferably about 2 to 6 hours. The conjugate thusobtained is dialyzed against water at about 0 to 18° C. in the routinemanner and stored frozen or optionally lyophilized and stored.

For the production of a polyclonal antibody, a warm-blooded animal isinoculated with the immunogen produced in the manner describedhereinbefore. The warm-blooded animal that can be used for this purposeincludes mammalian warm-blooded animals, e.g. rabbit, sheep, goat, rat,mouse, guinea pig, bovine, equine, swine, etc.; and avian species, e.g.chicken, dove, duck, goose, quail, etc. Regarding the methodology forinoculating a warm-blooded animal with the immunogen, the inoculum sizeof the immunogen may be just sufficient for antibody production. Forexample, the desired antibody can be produced in many instances byemulsifying 1 mg of the immunogen in 1 ml of saline with Freund'scomplete adjuvant and injecting the emulsion subcutaneously at the backand hind-limb footpad of rabbits 5 times at 4-week intervals. Forharvesting the antibody produced in the warm-blooded animal, for examplea rabbit, the blood is withdrawn from the auricular vein usually duringday 7 through day 12 after the last inoculation dose and centrifuged torecover an antiserum. For purification, the antiserum is generallysubjected to affinity chromatography using a carrier to which eachantigen peptide has been conjugated and the adsorbed fraction isrecovered to provide a polyclonal antibody.

The monoclonal antibody can be produced by the following method.

[Preparation of Monoclonal Antibody]

(a) Preparation of Monoclonal Antibody-Producing Cells.

The ligand polypeptide or G protein-coupled receptor protein isaministered to warm-blooded animals either solely or together withcarriers or diluents to the site where the production of antibody ispossible by the administration. In order to potentiate the antibodyproductivity upon the administration, complete Freund's adjuvants orincomplete Freund's adjuvants may be administered. The administration isusually carried out once every two to six weeks and two to ten times intotal. Examples of the applicable warm-blooded animals are monkeys,rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens andthe use of mice and rats is preferred.

In the preparation of the cells which produce monoclonal antibodies, ananimal wherein the antibody titer is noted is selected from warm-bloodedanimals (e.g. mice) immunized with antigens, then spleen or lymph nodeis collected after two to five days from the final immunization andantibody-producing cells contained therein are fused with myeloma cellsto give monoclonal antibody-producing hybridomas. Measurement of theantibody titer in antisera may, for example, be carried out by reactinga labeled ligand polypeptide or a labeled G protein-coupled receptorprotein (which will be mentioned later) with the antiserum followed bymeasuring the binding activity of the labeling agent with the antibody.The operation for fusing may be carried out, for example, by a method ofKoehler and Milstein (Nature, 256, 495, 1975), Examples of the fusionaccelerator are polyethylene glycol (PEG), Sendai virus, etc. and theuse of PEG is preferred.

Examples of the myeloma cells are NS-1, P3U1, SP2/0, AP-1, etc. and theuse of P3U1 is preferred. The preferred fusion ratio of the numbers ofantibody-producing cells used (spleen cells) to the numbers of myelomacells is within a range of about 1:1 to 20:1. When PEG (preferably, PEG1000 to PEG 6000) is added in a concentration of about 10-80% followedby incubating at 20-40° C. (preferably, at 30-37° C.) for one to tenminutes, an efficient cell fusion can be carried out.

Various methods may be applied for screening a hybridoma which producesanti-ligand polypeptide antibody or anti-G protein-coupled receptorantibody. For example, a supernatant liquid of hybridoma culture isadded to a solid phase (e.g. microplate) to which the ligand polypeptideantigen or the G protein-coupled receptor protein antigen is adsorbedeither directly or with a carrier, then anti-immunoglobulin antibody(anti-mouse immunoglobulin antibody is used when the cells used for thecell fusion are those of mouse) which is labeled with a radioactivesubstance, an enzyme or the like, or protein A is added thereto and thenanti-ligand polypeptide monoclonal antibodies or anti-G protein-coupledreceptor monoclonal antibodies bound on the solid phase are detected; ora supernatant liquid of the hybridoma culture is added to the solidphase to which anti-immunoglobulin or protein A is adsorbed, then theligand polypeptide or the G protein-coupled receptor labeled with aradioactive substance or an enzyme is added and anti-ligand polypeptideor anti-G protein-coupled receptor monoclonal antibodies bonded with thesolid phase is detected.

Selection and cloning of the anti-ligand polypeptide monoclonalantibody- or the anti-G protein-coupled receptor monoclonalantibody-producing hybridoma may be carried out by methods per se knownto those of skill in the art or methods similar thereto. Usually, it iscarried out in a medium for animal cells, containing HAT (hypoxanthine,aminopterin and thymidine). With respect to a medium for the selection,for the cloning and for the growth, any medium may be used so far ashybridoma is able to grow therein. Examples of the medium are an RPMI1640 medium (Dainippon Pharmaceutical Co., Ltd., Japan) containing 1-20%(preferably 10-20%) of fetal calf serum (FCS), a GIT medium (Wako PureChemical, Japan) containing 1-20% of fetal calf serum and a serum-freemedium for hybridoma culturing (SFM-101; Nissui Seiyaku, Japan). Theculturing temperature is usually 20-40° C. and, preferably, about 37° C.The culturing time is usually from five days to three weeks and,preferably, one to two weeks. The culturing is usually carried out in 5%carbon dioxide gas. The antibody titer of the supernatant liquid of thehybridoma culture may be measured by the same manner as in theabove-mentioned measurement of the antibody titer of the anti-ligandpolypeptide or the anti-G protein-coupled receptor in the antiserum.

(b) Purification of the Monoclonal Antibody.

Like in the separation/purification of conventional polyclonalantibodies, the separation/purification of the anti-ligand polypeptidemonoclonal antibody or the anti-G protein-coupled receptor monoclonalantibody may be carried out by methods for separating/purifyingimmunoglobulin such as salting-out, precipitation with an alcohol,isoelectric precipitation, electrophoresis, adsorption/deadsorptionusing ion exchangers such as DEAE, ultracentrifugation, gel filtration,specific purifying methods in which only an antibody is collected bytreatment with an active adsorbent such as an antigen-binding solidphase, protein A or protein G and the bond is dissociated whereupon theantibody is obtained.

The ligand polypeptide antibody or the G protein-coupled receptorantibody which is manufactured by the aforementioned method (a) or (b)is capable of specifically recognizing ligand polypeptide or Gprotein-coupled receptors and, accordingly, it can be used for aquantitative determination of the ligand polypeptide or the Gprotein-coupled receptor in test liquid samples and particularly for aquantitative determination by sandwich immunoassays.

Thus, the present invention provides, for example, the followingmethods:

-   (i) a quantitative determination of a ligand polypeptide or a G    protein-coupled receptor in a test liquid sample, which comprises-   (a) competitively reacting the test liquid sample and a labeled    ligand polypeptide or a labeled G protein-coupled receptor with an    antibody which reacts with the ligand polypeptide or the G    protein-coupled receptor, and-   (b) measuring the ratio of the labeled ligand polypeptide or the    labeled G protein-coupled receptor binding with said antibody; and    -   (ii) a quantitative determination of a ligand polypeptide or a G        protein-coupled receptor in a test liquid sample, which        comprises-   (a) reacting the test liquid sample with an antibody immobilized on    an insoluble carrier and a labeled antibody simultaneously or    continuously, and-   (b) measuring the activity of the labeling agent on the insoluble    carrier    wherein one antibody is capable of recognizing the N-terminal region    of the ligand polypeptide or the G protein-coupled receptor while    another antibody is capable of recognizing the C-terminal region of    the ligand polypeptide or the G protein-coupled receptor.

When the monoclonal antibody of the present invention recognizing aligand polypeptide or G protein-coupled receptor (hereinafter, may bereffered to as “anti-ligand polypeptide or anti-G protein-coupledreceptor antibody”) is used, ligand polypeptide or G protein-coupledreceptors can be measued and, moreover, can be detected by means of atissue staining, etc. as well. For such an object, antibody moleculesper se may be used or F(ab′)₂ Fab′ or Fab fractions of the antibodymolecule may be used too. There is no particular limitation for themeasuring method using the antibody of the present invention and anymeasuring method may be used so far as it relates to a method in whichthe amount of antibody, antigen or antibody-antigen complex, dependingon or corresponding to the amount of antigen, e.g. the amount of ligandpolypeptide or G protein-coupled receptor, etc. in the liquid sample tobe measured, is detected by a chemical or a physical means and thencalculated using a standard curve prepared by a standard solutioncontaining the known amount of antigen. For exmaple, nephrometry,competitive method, immunometric method and sanwich method are suitablyused and, in terms of sensitivity and specificity, the sandwich methodwhich will be described herein later is particularly preferred.

Examples of the labeling agent used in the measuring method using thelabeling substance are radioisotopes, enzymes, fluorescent substances,luminescent substances, colloids, magnetic substances, etc. Examples ofthe radioisotope are [¹²⁵ I], [¹³¹I], [³H] and [¹⁴C]; preferred examplesof the enzyme are those which are stable and with big specific activity,such as β-galactosidase, β-glucosidase, alkali phosphatase, peroxidaseand malate dehydrogenase; examples of the fluorescent substance arefluorescamine, fluorescein isothiocyanate, etc.; and examples of theluminescent substance are luminol, luminol derivatives, luciferin,lucigenin, etc. Further, a biotin-avidin system may also be used forbinding an antibody or antigen with a labeling agent.

In an insolubilization (immobilization) of antigens or antibodies, aphysical adsorption may be used or a chemical binding which is usuallyused for insolubilization or immobilization of proteins or enzymes maybe used as well. Examples of the carrier are insoluble polysaccharidessuch as agarose, dextran and cellulose; synthetic resins such aspolystyrene polyacrylamide and silicone; glass; etc.

In a sandwich (or two-site) method, the test liquid is made to reactwith an insolubilized anti-ligand polypeptide or anti-G protein-coupledreceptor antibody (the first reaction), then it is made to react with alabeled anti-ligand polypeptide or a labeled anti-G protein-coupledreceptor antibody (the second reaction) and the activity of the labelingagent on the insoluble carrier is measued whereupon the amount of theligand polypeptide or the G protein-coupled receptor in the test liquidcan be determined. The first reaction and the second reaction may beconducted reversely or simultaneously or they may be conducted with aninterval. The type of the labeling agent and the method ofinsolubilization (immobilization) may be the same as those mentionedalready herein. In the immunoassay by means of a sandwich method, it isnot always necessary that the antibody used for the labeled antibody andthe antibody for the solid phase is one type or one species but, with anobject of improving the measuring sensitivity, etc., a mixture of two ormore antibodies may be used too.

In the method of measuring ligand polypeptide or G protein-coupledreceptors by the sandwich method of the present invention, the preferredanti-ligand polypeptide antibodies or anti-G protein-coupled receptorantibodies used for the first and the second reactions are antibodieswherein their sites binding to the ligand polypeptide or the Gprotein-coupled receptors are different each other. Thus, the antibodiesused in the first and the second reactions are those wherein, when theantibody used in the second reaction recognizes the C-terminal region ofthe ligand polypeptide or the G protein-coupled receptor, then theantibody recognizing the site other than C-terminal regions, e.g.recognizing the N-terminal region, is preferably used in the firstreaction.

The anti-ligand polypeptide antibody or the anti-G protein-coupledreceptor antibody of the present invention may be used in a measuringsystem other than the sandwich method such as a competitive method, animmunometric method and a naphrometry. In a competitive method, anantigen in the test solution and a labeled antigen are made to reactwith an antibody in a competitive manner, then an unreacted labeledantigen (F) and a labeled antigen binding with an antibody (B) areseparated (i.e. B/F separation) and the labeled amount of any of B and Fis measured whereupon the amount of the antigen in the test solution isdetermined. With respect to a method for such a reaction, there are aliquid phase method in which a soluble antibody is used as the antibodyand the B/F separation is conducted by polyethylene glycol, a secondantibody to the above-mentioned antibody, etc.; and a solid phase methodin which an immobilized antibody is used as the first antibody or asoluble antibody is used as the first antibody while an immobilizedantibody is used as the second antibody.

In an immunometric method, an antigen in the test solution and animmobilized antigen are subjected to a competitive reaction with acertain amount of a labeled antibody followed by separating into solidand liquid phases; or the antigen in the test solution and an excessamount of labeled antibody are made to react, then a immobilized antigenis added to bind an unreacted labeled antibody with the solid phase andseparated into solid and liquid phases. After that, the labeled amountof any of the phases is measured to determine the antigen amount in thetest solution.

In a nephrometry, the amount of insoluble sediment which is produced asa result of the antigen-antibody reaction in a gel or in a solution ismeasured. Even when the antigen amount in the test solution is small andonly a small amount of the sediment is obtained, a laser nephrometrywherein scattering of laser is utilized can be suitably used.

In applying each of those immunological measuring methods (immunoassays)to the measuring method of the present invention, it is not necessary toset up any special condition, operation, etc. therefor. A measuringsystem (assay system) for ligand polypeptide or G protein-coupledreceptor may be constructed taking the technical consideration of thepersons skilled in the art into consideration in the conventionalconditions and operations for each of the methods. With details of thoseconventional technical means, a variety of reviews, reference books,etc. may be referred to. They are, for example, Hiroshi Irie (ed):“Radioimmunoassay” (Kodansha, Japan, 1974); Hiroshi Irie (ed):“Radioimmunoassay; Second Series” (Kodansha, Japan, 1979); Eiji Ishikawaet al. (ed): “Enzyme Immunoassay” (Igaku Shoin, Japan, 1978); EijiIshikawa et al. (ed): “Enzyme-Immunoassay” (Second Edition) (IgakuShoin, Japan, 1982); Eiji Ishikawa et al. (ed): “Enzyme Immunoassay”(Third Edition) (Igaku Shoin, Japan, 1987); “Methods in Enzymology” Vol.70 (Immunochemical Techniques (Part A)); ibid. Vo. 73 (ImmunochemicalTechniques (Part B)); ibid. Vo. 74 (Immunochemical Techniques (Part C));ibid. Vo. 84 (Immunochemical Techniques (Part D: SelectedImmunoassays)); ibid. Vol. 92 (Immunochemical Techniques (Part E:Monoclonal Antibodies and General Immunoassay Methods)); ibid. Vol. 121(Immunochemical Techniques (Part I: Hybridoma Technology and MonoclonalAntibodies)) (Academic Press); etc.

As such, the amount of ligand polypeptide or G protein-coupled receptorproteins can now be determined with a high precision using theanti-ligand polypeptide or the anti-G protein-coupled receptor antibodyof the present invention.

In the specification and drawings of the present application, theabbreviations used for bases (nucleotides), amino acids and so forth arethose recommended by the IUPAC-IUB Commission on BiochemicalNomenclature or those conventionally used in the art. Examples thereofare given below. Amino acids for which optical isomerism is possibleare, unless otherwise specified, in the L form.

-   DNA: Deoxyribonucleic acid-   cDNA: Complementary deoxyribonucleic acid-   A: Adenine-   T: Thymine-   G: Guanine-   C: Cytosine-   RNA: Ribonucleic acid-   mRNA: Messenger ribonucleic acid-   DATP: Deoxyadenosine triphosphate-   dTTP: Deoxythymidine triphosphate-   dGTP: Deoxyguanosine triphosphate-   dCTP: Deoxycytidine triphosphate-   ATP: Adenosine triphosphate-   EDTA: Ethylenediamine tetraacetic acid-   SDS: Sodium dodecyl sulfate-   EIA: Enzyme Immunoassay    -   G, Gly: Glycine (or Glycyl)    -   A, Ala: Alanine (or Alanyl)    -   V, Val: Valine (or Valyl)    -   L, Leu: Leucine (or Leucyl)    -   I, Ile: Isoleucine (or Isoleucyl)    -   S, Ser: Serine (or Seryl)    -   T, Thr: Threonine (or Threonyl)    -   C, Cys: Cysteine (or Cysteinyl)    -   M, Met: Methionine (or Methionyl)    -   E, Glu: Glutamic acid (or Glutamyl)    -   D, Asp: Aspartic acid (or Aspartyl)    -   K, Lys: Lysine (or Lysyl)    -   R, Arg: Arginine (or Arginyl)    -   H, His: Histidine (or Histidyl)    -   F, Phe: Phenylalamine (or Phenylalanyl)    -   Y, Tyr: Tyrossine (or Tyrosyl)    -   W, Trp: Tryptophan (or Tryptophanyl)    -   P, Pro: Proline (or Prolyl)    -   N, Asn: Asparagine (or Asparaginyl)    -   Q, Gln: Glutamine (or Glutaminyl)    -   pGlu: Pyroglutamic acid (or Pyroglutamyl)    -   Me: Methyl    -   Et: Ethyl    -   Bu: Butyl    -   Ph: Phenyl    -   TC: Thiazolidinyl-4(R)-carboxamide

In this specification, substitutions, protective groups and reagentscommonly used are indicated by the following abbreviations:

-   -   BHA: benzhydrylamine    -   PMBHA: p-methylbenzhydrylamine    -   Tos: p-toluenesulfonyl    -   CHO: formyl    -   HONB: N-hydroxy-5-norbornene-2,3-dicarboxyimide    -   OcHex: cyclohexyl ester    -   Bzl: benzyl    -   Bom: benzyloxymethyl    -   Br-Z: 2-bromobenzyloxycarbonyl    -   Boc: t-butoxycarbonyl    -   DCM: dichloromethane    -   HOBt: 1-hydroxybenztriazole    -   DCC: N,N′-dicyclohexylcarbodiimide    -   TFA: trifluoro acetate    -   DIEA: diisopropylethylamine    -   Fmoc: N-9-fluorenylmethoxycarbonyl    -   DNP: dinitrophenyl    -   Bum: t-butoxymethyl    -   Trt: trityl

Each SEQ ID NO set forth in the SEQUENCE LISTING of the specificationrefers to the following sequence:

-   [SEQ ID NO:1] is an entire amino acid sequence of the bovine    pituitary-derived ligand polypeptide encoded by the cDNA included in    pBOV3.-   [SEQ ID NO:2] is an entire nucleotide sequence of the bovine    pituitary-derived ligand polypeptide cDNA.-   [SEQ ID NO:3] is an amino acid sequence of the bovine    pituitary-derived ligand polypeptide which was obtained by    purification and analysis of N-terminal sequence for P-3 fraction.    The amino acid sequence corresponds to 23rd to 51st positions of the    amino acid sequence of SEQ ID NO:1.-   [SEQ ID NO:4] is an amino acid sequence of the bovine    pituitary-derived ligand polypeptide which was obtained by    purification and analysis of N-terminal sequence for P-2 fraction.    The amino acid sequence corresponds to 34th to 52nd positions of the    amino acid sequencce of SEQ ID NO:1.-   [SEQ ID NO:5] is an amino acid sequence of the bovine    pituitary-derived ligand polypeptide. The amino acid sequence    corresponds to 23rd to 53rd positions of the amino acid sequence of    SEQ ID NO:1.-   [SEQ ID NO:6] is an amino acid sequence of the bovine    pituitary-derived ligand polypeptide. The amino acid sequence    corresponds to 23rd to 54th positions of the amino acid sequence of    SEQ ID NO:1.-   [SEQ ID NO:7] is an amino acid sequence of the bovine    pituitary-derived ligand polypeptide. The amino acid sequence    corresponds to 23rd to 55th positions of the amino acid sequence of    SEQ ID NO:1.-   [SEQ ID NO:8] is an amino acid sequence of the bovine    pituitary-derived ligand polypeptide. The amino acid sequence    corresponds to 34th to 53rd positions of the amino acid sequence of    SEQ ID NO:1.-   [SEQ ID NO:9] is an amino acid sequence of the bovine    pituitary-derived ligand polypeptide. The amino acid sequence    corresponds to 34th to 54th positions of the amino acid sequence of    SEQ ID NO:1.-   [SEQ ID NO:10] is an amino acid sequence of the bovine    pituitary-derived ligand polypeptide. The amino acid sequence    corresponds to 34th to 55th positions of the amino acid sequence of    SEQ ID NO:1.-   [SEQ ID NO:11] is a nucleotide sequence of DNA coding for the bovine    pituitary-derived ligand polypeptide (SEQ ID NO:3).-   [SEQ ID NO:12] is a nucleotide sequence of DNA coding for the bovine    pituitary-derived ligand polypeptide (SEQ ID NO:4).-   [SEQ ID NO:13] is a nucleotide sequence of DNA coding for the bovine    pituitary-derived ligand polypeptide (SEQ ID NO:5).-   [SEQ ID NO:14] is a nucleotide sequence of DNA coding for the bovine    pituitary-derived ligand polypeptide (SEQ ID NO:6).-   [SEQ ID NO:15] is a nucleotide sequence of DNA coding for the bovine    pituitary-derived ligand polypeptide (SEQ ID NO:7).-   [SEQ ID NO:16] is a nucleotide sequence of DNA coding for the bovine    pituitary-derived ligand polypeptide (SEQ ID NO:8).-   [SEQ ID NO:17] is a nucleotide sequence of DNA coding for the bovine    pituitary derived ligand polypeptide (SEQ ID NO:9).-   [SEQ ID NO:18] is a nucleotide sequence of DNA coding for the bovine    pituitary-derived ligand polypeptide (SEQ ID NO:10).-   [SEQ ID NO:19] is a partial amino acid sequence of the human    pituitary-derived G protein-coupled receptor protein encoded by the    human pituitary-derived G protein-coupled receptor protein cDNA    fragment included in p19P2.-   [SEQ ID NO:20] is a partial amino acid sequence of the human    pituitary-derived G protein-coupled receptor protein encoded by the    human pituitary-derived G protein-coupled receptor protein cDNA    fragment include in p19P2.-   [SEQ ID NO:21] is an entire amino acid sequence of the human    pituitary-derived G protein-coupled receptor protein encoded by the    human pituitary-derived G protein-coupled receptor protein cDNA    include in phGR3.-   [SEQ ID NO:22] is a partial amino acid sequence of the mouse    pancreatic β-cell line, MIN6-derived G protein-coupled receptor    protein encoded by the mouse pancreatic β-cell line, MIN6-derived G    protein-coupled receptor protein cDNA fragment having a nucleotide    sequence (SEQ ID NO:27), derived based upon the nucleotide sequences    of the mouse pancreatic β-cell line, MIN6-derived G protein-coupled    receptor protein cDNA fragments each included in pG3-2 and pG1-10.-   [SEQ ID NO:23] is a partial amino acid sequence of the mouse    pancreatic β-cell line, MIN6-derived G protein-coupled receptor    protein encoded by p5S38.-   [SEQ ID NO:24] is a nucleotide sequence of the human    pituitary-derived G protein-coupled receptor protein cDNA fragment    include in p19P2.-   [SEQ ID NO:25] is a nucleotide sequence of the human    pituitary-derived G protein-coupled receptor protein cDNA fragment    include in p19P2.-   [SEQ ID NO:26] is an entire nucleotide sequence of the human    pituitary-derived G protein-coupled receptor protein cDNa include in    phGR3.-   [SEQ ID NO:27] is a nucleotide sequence of the mouse pancreatic    β-cell line, MIN6-derived G protein-coupled receptor protein cDNA,    derived based upon the nucleotide sequences of the mouse pancreatic    β-cell line, MIN6-derived G protein-coupled receptor protein cDNA    fragments each included in pG3-2 and pG1-10.-   [SEQ ID NO: 28] is a nucleotide sequence of the mouse pancreatic    β-cell line, MIN6-derived G protein-coupled receptor protein cDNA    include in p5S38.-   [SEQ ID NO:29] is a synthetic DNA primer for screening of cDNA    coding for the G protein-coupled receptor protein.-   [SEQ ID NO:30] is a synthetic DNA primer for screening of cDNA    coding for the G protein-coupled receptor protein.-   [SEQ ID NO:31] is a synthetic DNA primer for screening of cDNA    coding for the G protein-coupled receptor protein.-   [SEQ ID NO:32] is a synthetic DNA primer for screening of cDNA    coding for the G protein-coupled receptor protein.-   [SEQ ID NO:33] is a synthetic DNA primer for screening of cDNA    coding for the G protein-coupled receptor protein.-   [SEQ ID NO:34] is a synthetic DNA primer for screening of cDNA    coding for the G protein-coupled receptor protein.-   [SEQ ID NO:35] is a synthetic DNA primer for screening of cDNA    coding for the bovine pituitary-derived ligand polypeptide, wherein    the primer is represented by P5-1.-   [SEQ ID NO:36] is a synthetic DNA primer for screening of cDNA    coding for the bovine pituitary-derived ligand polypeptide, wherein    the primer is represented by P3-1.-   [SEQ ID NO:37] is a synthetic DNA primer for screening of cDNA    coding for the bovine pituitary-derived ligand polypeptide, wherein    the primer is represented by P3-2.-   [SEQ ID NO:38] is a synthetic DNA primer for screening of cDNA    coding for the bovine pituitary-derived ligand polypeptide, wherein    the primer is represented by PE.-   [SEQ ID NO:39] is a synthetic DNA primer for screening of cDNA    coding for the bovine pituitary-derived ligand polypeptide, wherein    the primer is represented by PDN.-   [SEQ ID NO:40] is a synthetic DNA primer for screening of cDNA    coding for the bovine pituitary-derived ligand polypeptide, wherein    the primer is represented by FB.-   [SEQ ID NO:41] is a synthetic DNA primer for screening of cDNA    coding for the bovine pituitary-derived ligand polypeptide, wherein    the primer is represented by FC.-   [SEQ ID NO:42] is a synthetic DNA primer for screening of cDNA    coding for the bovine-pituitary-derived ligand polypeptide, wherein    the primer is represented by BOVF.-   [SEQ ID NO:43] is a synthetic DNA primer for screening of cDNA    coding for the bovine pituitary-derived ligand polypeptide, wherein    the primer is represented by BOVR.-   [SEQ ID NO:44] is an entire amino acid sequence of the bovine    genome-derived ligand polypeptide.-   [SEQ ID NO: 45] is an entire amino acid sequence of the rat type    ligand polypeptide encoded by the cDNA included in pRAV3.-   [SEQ ID NO:46] is an entire nucleotide sequence of the rat type    ligand polypeptide cDNA.-   [SEQ ID NO:47] is an amino acid sequence of the rat type ligand    polypeptide. The amino acid sequence corresponds to 22nd to 52nd    positions of the amino acid sequence of SEQ ID NO:45.-   [SEQ ID NO:48] is an amino acid sequence of the rat type ligand    polypeptide. The amino acid sequence corresponds to 22nd to 53rd    positions of the amino acid sequence of SEQ ID NO:45.-   [SEQ ID NO:49] is an amino acid sequence of the rat type ligand    polypeptide. The amino acid sequence corresponds to 22nd to 54th    positions of the amino acid sequence of SEQ ID NO:45.-   [SEQ ID NO:50] is an amino acid sequence of the rat type ligand    polypeptide. The amino acid sequence corresponds to 33rd to 52nd    positions of the amino acid sequence of SEQ ID NO:45.-   [SEQ ID NO:51] is an amino acid sequence of the rat type ligand    polypeptide. The amino acid sequence corresponds to 33rd to 53rd    positions of the amino acid sequence of SEQ ID NO:45.-   [SEQ ID NO:52] is an amino acid sequence of the rat type ligand    polypeptide. The amino acid sequence corresponds to 33rd to 54th    positions of the amino acid sequence of SEQ ID NO.45.-   [SEQ ID NO:53] is a nucleotide sequence encoding for the rat type    ligand polypeptide of SEQ ID NO:47.-   [SEQ ID NO:54] is a nucleotide sequence encoding for the rat type    ligand polypeptide of SEQ ID NO:48.-   [SEQ ID NO:55] is a nucleotide sequence encoding for the rat type    ligand polypeptide of SEQ ID NO:49.-   [SEQ ID NO:56] is a nucleotide sequence encoding for the rat type    ligand polypeptide of SEQ ID NO:50.-   [SEQ ID NO:57] is a nucleotide sequence encoding for the rat type    ligand polypeptide of SEQ ID NO:51.-   [SEQ ID NO:58] is a nucleotide sequence encoding for the rat type    ligand polypeptide of SEQ ID NO:52.-   [SEQ ID NO:59] is an entire amino acid sequence of the human type    ligand polypeptide encoded by the cDNA included in pHOB7.-   [SEQ ID NO:60] is an entire nucleotide sequence of the human type    ligand polypeptide cDNA.-   [SEQ ID NO:61] is an amino acid sequence of the human type ligand    polypeptide. The amino acid sequence corresponds to 23rd to 53rd    positions of the amino acid sequence of SEQ ID NO.59.-   [SEQ ID NO:62] is an amino acid sequence of the human type ligand    polypeptide. The amino acid sequence corresponds to 23rd to 54th    positions of the amino acid sequence of SEQ ID NO.59.-   [SEQ ID NO:63] is an amino acid sequence of the human type ligand    polypeptide. The amino acid sequence corresponds to 23rd to 55th    positions of the amino acid sequence of SEQ ID NO.59.-   [SEQ ID NO:64] is an amino acid sequence of the human type ligand    polypeptide. The amino acid sequence corresponds to 34th to 53rd    positions of the amino acid sequence of SEQ ID NO.59.-   [SEQ ID NO:65] is an amino acid sequence of the human type ligand    polypeptide. The amino acid sequence corresponds to 34th to 54th    positions of the amino acid sequence of SEQ ID NO.59.-   [SEQ ID NO:66] is an amino acid sequence of the human type ligand    polypeptide. The amino acid sequence corresponds to 34th to 55th    positions of the amino acid sequence of SEQ ID NO.59.-   [SEQ ID NO:67] is a nucleotide sequence encoding for the human type    ligand polypeptide of SEQ ID NO:61.-   [SEQ ID NO:68] is a nucleotide sequence encoding for the human type    ligand polypeptide of SEQ ID NO:62.-   [SEQ ID NO:69] is a nucleotide sequence encoding for the human type    ligand polypeptide of SEQ ID NO:63.-   [SEQ ID NO:70] is a nucleotide sequence encoding for the human type    ligand polypeptide of SEQ ID NO:64.-   [SEQ ID NO:71] is a nucleotide sequence encoding for the human type    ligand polypeptide of SEQ ID NO:65.-   [SEQ ID NO:72] is a nucleotide sequence encoding for the human type    ligand polypeptide of SEQ ID NO:66.-   [SEQ ID NO:73] is a partial amino acid sequence of the ligand    polypeptide, wherein Xaa of the 10th position is Ala or Thr, Xaa of    the 11th position is Gly or Ser and Xaa of the 21st position is H,    Gly or GlyArg.-   [SEQ ID NO:74] is a partial amino acid sequence of the ligand    polypeptide, wherein Xaa of the 3rd position is Ala or Thr, Xaa of    the 5th position is Gln or Arg and Xaa of the 10th position is Ile    or Thr.-   [SEQ ID NO:75] is a synthetic DNA primer for screening of cDNA    coding for the rat type ligand polypeptide, wherein the primer is    represented by RA.-   [SEQ ID NO:76] is a synthetic DNA primer for screening of cDNA    coding for the rat type ligand polypeptide, wherein the primer is    represented by RC.-   [SEQ ID NO:77] is a synthetic DNA primer for screening of cDNA    coding for the rat type ligand polypeptide, wherein the primer is    represented by rF.-   [SEQ ID NO:78] is a synthetic DNA primer for screening of cDNA    coding for the rat type ligand polypeptide, wherein the primer is    represented by rR.-   [SEQ ID NO:79] is a synthetic DNA primer for screening of cDNA    coding for the human type ligand polypeptide, wherein the primer is    represented by R1.-   [SEQ ID NO:80] is a synthetic DNA primer for screening of cDNA    coding for the human type ligand polypeptide, wherein the primer is    represented by R3.-   [SEQ ID NO:81] is a synthetic DNA primer for screening of cDNA    coding for the human type ligand polypeptide, wherein the primer is    represented by R4.-   [SEQ ID NO:82] is a synthetic DNA primer for screening of cDNA    coding for the human type ligand polypeptide, wherein the primer is    represented by HA.-   [SEQ ID NO:83] is a synthetic DNA primer for screening of cDNA    coding for the human type ligand polypeptide, wherein the primer is    represented by HB.-   [SEQ ID NO:84] is a synthetic DNA primer for screening of cDNA    coding for the human type ligand polypeptide, wherein the primer is    represented by HE.-   [SEQ ID NO:85] is a synthetic DNA primer for screening of cDNA    coding for the human type ligand polypeptide, wherein the primer is    represented by HF.-   [SEQ ID NO:86] is a synthetic DNA primer for screening of cDNA    coding for the human type ligand polypeptide, wherein the primer is    represented by 5H.-   [SEQ ID NO:87] is a synthetic DNA primer for screening of cDNA    coding for the human type ligand polypeptide, wherein the primer is    represented by 3HN.-   [SEQ ID NO:88] is a synthetic DNA primer for screening of cDNA    coding for the rat type G protein-coupled receptor protein (UHR-1),    wherein the primer is represented by rRECF.-   [SEQ ID NO:89] is a synthetic DNA primer for screening of cDNA    coding for the rat type G protein-coupled receptor protein (UHR-1),    wherein the primer is represented by rRECR.-   [SEQ ID NO:90] is a synthetic DNA which is used for amplification of    G3PDH, UHR-1 and ligand, wherein the primer represented by r19F.-   [SEQ ID NO:91] is a synthetic DNA which is used for amplification of    G3PDH, UHR-1 and ligand, wherein the primer represented by r19R.-   [SEQ ID NO:92] is a N-terminal peptide of the ligand polypeptide,    which is used for antigen. (Peptide-I)-   [SEQ ID NO:93] is a C-terminal peptide of the ligand polypeptide,    which is used for antigen. (Peptide-II)-   [SEQ ID NO:94] is a peptide of the central portion in ligand    polypeptide, which is used for antigen. (Peptide-III)-   [SEQ ID NO:95] is a synthetic DNA primer for screening of cDNA    coding for rat type G protein-coupled receptor protein (UHR-1).-   [SEQ ID NO:96] is a synthetic DNA primer for screening of cDNA    coding for rat type G protein-coupled receptor protein (UHR-1).

The transformant Escherichia coli, designated INVαF′/p19P2, which isobtained in the Example 2 mentioned herein below, is on deposit underthe terms of the Budapest Treaty from Aug. 9, 1994, with the NationalInstitute of Bioscience and Human-Technology (NIBH), Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry, Japan and has been assigned the Accession Number FERM BP-4776.It is also on deposit from Aug. 22, 1994 with the Institute forFermentation, Osaka, Japan (IFO) and has been assigned the AccessionNumber IFO 15739.

The transformant Escherichia coli, designated INVαF′/pG3-2, which isobtained in the Example 4 mentioned herein below, is on deposit underthe terms of the Budapest Treaty from Aug. 9, 1994, with NIBH and hasbeen assigned the Accession Number FERM BP-4775. It is also on depositfrom Aug. 22, 1994 with IFO and has been assigned the Accession NumberIFO 15740.

The transformant Escherichia coli, designated JM109/phGR3, which isobtained in the Example 5 mentioned herein below, is on deposit underthe terms of the Budapest Treaty from Sep. 27, 1994, with NIBH and hasbeen assigned the Accession Number FERM BP-4807. It is also on depositfrom Sep. 22, 1994 with IFO and has been assigned the Accession NumberIFO 15748.

The transformant Escherichia coli, designated JM109/p5S38, which isobtained in the Example 8 mentioned herein below, is on deposit underthe terms of the Budapest Treaty from Oct. 27, 1994, with NIBH and hasbeen assigned the Accession Number FERM BP-4856. It is also on depositfrom Oct. 25, 1994 with IFO and has been assigned the Accession NumberIFO 15754.

The transformant Escherichia coli, designated JM109/pBOV3, which isobtained in the Example 20 mentioned herein below, is on deposit underthe terms of the Budapest Treaty from Feb. 13, 1996, with NIBH and hasbeen assigned the Accession Number FERM BP-5391. It is also on depositfrom Jan. 25, 1996 with IFO and has been assigned the Accession NumberIFO 15910.

The transformant Escherichia coli, designated JM109/pRAV3, which isobtained in the Example 29 mentioned herein below, is on deposit underthe terms of the Budapest Treaty from Sep. 12, 1996, with NIBH and hasbeen assigned the Accession Number FERM BP-5665. It is also on depositfrom Sep. 3, 1996 with IFO and has been assigned the Accession NumberIFO 16012.

The transformant Escherichia coli, designated JM109/pHOV7, which isobtained in the Example 32 mentioned herein below, is on deposit underthe terms of the Budapest Treaty from Sep. 12, 1996, with NIBH and hasbeen assigned the Accession Number FERM BP-5666. It is also on depositfrom Sep. 5, 1996 with IFO and has been assigned the Accession NumberIFO 16013.

Industrial Application

The bioactive substance of the present invention, namely the ligandpolypeptide or its amide or ester thereof, or a salt thereof, a partialpeptide thereof, or the DNA coding for said ligand polypeptide, hasfunction modulating activity for various tissues or internal organs,e.g. heart, lung, liver, spleen, thymus, kidney, adrenal glands, skeltalmuscle, testis etc., besides pituitary, central nervous system orpancreas, and are useful as medicines. Furthermore, the substance isuseful for the screening of agonists or antagonists of G protein-coupledreceptor proteins. The compounds which can be obtained by such screeningalso have function modulating activity for above-described tissues orinternal organs, and are useful as medicines.

EXAMPLES

Described below are working examples of the present invention which areprovided only for illustrative purposes, and not to limit the scope ofthe present invention.

REFERENCE EXAMPLE 1

Preparation of Synthetic DNA Primer for Amplifying DNA Coding for Gprotein-coupled receptor Protein

A compariton of deoxyribonucleotide sequences coding for the known aminoacid sequences corresponding to or near the first membrane-spanningdomain each of human-derived TRH receptor protein (HTRHR), human-derivedRANTES receptor protein (L10918, HUMRANTES), human Burkitt'slymphoma-derived unknown ligand receptor protein (X68149, HSBLR1A),human-derived somatostatin receptor protein (L14856, HUMSOMAT),rat-derived μ-opioid receptor protein (U02083, RNU02083), rat-derivedK-opioid receptor protein (U00442, U00442), human-derived neuromedin Breceptor protein (M734.82, HUMNMBR), human-derived muscarinicacetylcholine receptor protein (X15266, HSHM4), rat-derived adrenalineα₁B receptor protein (L08609, RATAADRE01), human-derived somatostatin 3receptor protein (M96738, HUMSSTR3X), human-derived C₅a receptor protein(HUMC5AAR), human-derived unknown ligand receptor protein (HUMRDC1A),human-derived unknown ligand receptor protein (M84605, HUMOPIODRE) andrat-derived adrenaline α₂B receptor protein (M91466, RATA2BAR) was made.As a result, highly homologous regions or parts were found.

Further, a comparison of deoxynucleotide sequences coding for the knownamino acid sequences corresponding to or near the sixthmembrane-spanning domain each of mouse-derived unknown ligand receptorprotein (M80481, MUSGIR), human-derived bombesin receptor protein(L08893, HUMBOMB3S), human-derived adenosine A2 receptor protein(S46950, S46950), mouse-derived unknown ligand receptor protein (D21061,MUSGPCR), mouse-derived TRH receptor protein (S43387, S43387),rat-derived neuromedin K receptor protein (J05189, RATNEURA),rat-derived adenosine A1 receptor protein. (M69045, RATA1ARA),human-derived neurokinin A receptor protein (M57414, HUMNEKAR),rat-derived adenosine A3 receptor protein (M94152, DATADENREC),human-derived somatostatin 1 receptor protein (M81829, HUMSRI1A),human-derived neurokinin 3 receptor protein (S86390, S86371S4),rat-derived unknown ligand receptor protein (X61496, RNCGPCR),human-derived somatostatin 4 receptor protein (L07061, HUMSSTR4Z) andrat-derived GnRH receptor protein (M31670, RATGNRHA) was made. As aresult, highly homologous regions or parts were found.

The aforementioned abbreviations in the parentheses are identifiers(reference numbers) which are indicated when GenBank/EMBL Data Bank isretrieved by using DNASIS Gene/Protein Sequencing Data Base (CD019,Hitachi Software Engineering, Japan) and are usually called “AccessionNumbers” or “Entry Names”. HTRHR is, however, the sequence as disclosedin Japanese Patent Publication No. 304797/1993 (EPA 638645).

Specifically, it was planned to incorporate mixed bases relying upon thebase regions that were in agreement with cDNAs coding for a large numberof receptor proteins in order to enhance base agreement of sequenceswith as many receptor cDNAs as possible even in other regions. Basedupon these sequences, the degenerate synthetic DNA having a nucleotidesequence represented by SEQ ID NO:29 or SEQ ID NO:30 which iscomplementary to the homologous nucleotide sequence were produced.[Synthetic DNAs] 5′-CGTGG (G or C) C (A or C) T (SEQ ID NO: 29) (G or C)(G or C) TGGGCAAC (A, G, C or T) (C or T) CCTG-3′ 5′-GT (A, G, C or T) G(A or T) (SEQ ID NO: 30) (A or G) (A or G) GGCA (A, G, C or T) CCAGCAGA(G or T) GGCAAA-3′

The parentheses indicate the incorporation of a plurality of bases,leading to multiple oligonucleotides in the primer preparation. In otherwords, nucleotide resides in parentheses of the aforementioned DNAs wereincorporated in the presence of a mixture of plural bases at the time ofsynthesis.

Example 1

Amplification of Receptor cDNA by PCR Using Human PituitaryGland-Derived cDNA

By using human pituitary gland-derived cDNA (QuickClone, CLONTECHLaboratories, Inc.) as a template, PCR amplification using the DNAprimers synthesized in Reference Example 1 was carried out. Thecomposition of the reaction solution consisted of the synthetic DNAprimers (SEQ: 5′ primer sequence and 3′ primer sequence) each in anamount of 1 μM, 1 ng of the template cDNA, 0.25 mM dNTPs, 1 μl of TaqDNA polymerase and a buffer attached to the enzyme kit, and the totalamount of the reaction solution was made to be 100 μl. The cycle foramplification including 95° C. for 1 min., 55° C. for 1 min. and 72° C.for 1 min. was repeated 30 times by using a Thermal Cycler (Perkin-ElmerCo.). Prior to adding Taq DNA polymerase, the remaining reactionsolution was mixed and was heated at 95° C. for 5 minutes and at 65° C.for 5 minutes. The amplified products were confirmed relying upon 1.2%agarose gel electrophoresis and ethidium bromide staining.

Example 2

Subcloning of PCR Product into Plasmid Vector and Selection of NovelReceptor Candidate Clone Via Decoding Nucleotide Sequence of InsertedcDNA Region

The PCR products were separated by using a 0.8% low-melting temperatureagarose gel, the band parts were excised from the gel with a razorblade, and were heat-melted, extracted with phenol and precipitated inethanol to recover DNAs. According to the protocol attached to a TACloning Kit (Invitrogen Co.), the recovered DNAs were subcloned into theplasmid vector, pCR™II (TM represents registered trademark). Therecombinant vectors were introduced into E. coli INVαF′ competent cells(Invitrogen Co.) to produce transformants. Then, transformant cloneshaving a cDNA-inserted fragment were selected in an LB agar culturemedium containing ampicillin and X-gal. Only transformant clonesexhibiting white color were picked with a sterilized toothstick toobtain transformant Escherichia coli INVαF′/p19P2.

The individual clones were cultured overnight in an LB culture mediumcontaining ampicillin and treated with an automatic plasmid extractingmachine (Kurabo Co., Japan) to prepare plasmid DNAS. An aliquot of theDNA thus prepared was cut by EcoRI to confirm the size of the cDNAfragment that was inserted. An aliquot of the remaining DNA was furtherprocessed with RNase, extracted with phenol/chloroform, and precipitatedin ethanol so as to be condensed. Sequencing was carried out by using aDyeDeoxy terminator cycle sequencing kit (ABI Co.), the DNAs weredecoded by using a fluorescent automatic sequencer, and the data of thenucleotide sequences obtained were read by using DNASIS (Hitachi SystemEngineering Co., Japan). The underlined portions represent regionscorresponding to the synthetic primers.

Homology retrieval was carried out based upon the determined nucleotidesequences [SEQ ID NO:24 and 25 (Here, the determined nucleotide sequenceis the nucleotide sequence which the underlined portion is deleted fromthe sequence of FIG. 1 or FIG. 2 respectively)].

As a result, it was learned that a novel G protein-coupled receptorprotein was encoded by the cDNA fragment insert in the plasmid, p19P2,possessed by the transformant Escherichia coli INVαF′/p19P2. To furtherconfirm this fact, by using DNASIS (Hitachi System Engineering Co.,Japan) the nucleotide sequences were converted into amino acid sequences[SEQ ID NO:19 and 20], and homology retrieval was carried out in view ofhydrophobicity plotting [FIGS. 3 and 4] and at the amino acid sequencelevel to find homology relative to neuropeptide Y receptor proteins[FIG. 5].

Example 3

Preparation of Poly(A)⁺RNA Fraction from Mouse Pancreatic β-Cell Strain,MIN6 and Synthesis of cDNA

A total RNA was prepared from the mouse pancreatic β-cell strain, MIN6(Jun-ichi Miyazaki et al., Endocrinology, Vol. 127, No. 1, p. 126-132)according to the guanidine thiocyanate method (Kaplan B. B. et al.,Biochem. J., 183, 181-184 (1979) and, then, poly(A)⁺RNA fractions wereprepared with a mRNA purifying kit (Pharmacia Co.). Next, to 5 μg of thepoly(A)⁺RNA fraction was added a random DNA hexamer (BRL Co.) as aprimer, and the resulting mixture was subjected to reaction with mouseMoloney Leukemia virus (MMLV) reverse transcriptase (BRL Co.) in thebuffer attached to the MMLV reverse transcriptase kit to synthesizecomplementary DNAs. The reaction product was extracted withphenol/chloroform (1:1), precipitated in ethanol, and was then dissolvedin 30 μl of TE buffer (10 mM Tris-HCL at pH8.0, 1 mM EDTA at pH8.0).

Example 4

Amplification of Receptor cDNA by PCR Using MIN6-Derived cDNA andSequencing

By suing, as a template, 5 μl of cDNA prepared from the mouse pancreaticβ-cell strain, MIN6 in the above Example 3, PCR amplification using theDNA primers synthesized in Reference Example 1 was carried out under thesame condition as in Example 1. The resulting PCR product was subclonedinto the plasmid vector, pCR™II, in the same manner as in Example 2 toobtain a plasmid, pG3-2. The plasmid pG3-2 was transfected into E. coliINVαF′ to obtain transformed Escherichia coli INVαF′/pG3-2.

By using, as a template, 5 μl of the cDNA parepared from the mousepancreatic β-cell strain, MIN6, PCR amplification using DNA primers asdisclosed in Libert F. et al., “Science, 244: 569-572, 1989”, i.e., adegenerate synthetic primer represented by the following sequence:5′-CTGTG (C or T) G (C or T) (G (SEQ ID NO: 31) or C) AT (C or T) GCIIT(G or T) GA (C or T) (A or C) G (G or C) TAC-3′

-   wherein I is inosine; and

a degenerate synthetic primer represented by the following sequence:5′-A (G or T) G (A or T) AG (A or (SEQ ID NO: 32) T) AGGGCAGCCAGCAGAI (Gor C) (A or G) (C or T) GAA-3′

-   wherein I is inosine,    was carried out under the same conditions as in Working Example 1.    The resulting PCR product was subcloned into the plasmid vector,    pCR™II, in the same manner as described in Example 2 to obtain a    plasmid, pG1-10.

The reaction for determining the nucleotide sequence (sequencing) wascarried out with a DyeDeoxy terminator cycle sequencing kit (ABI Co.),the DNA was decoded with the fluorescent automatic sequencer (ABI Co.),and the data of the nucleotide sequence obtained were analyzed withDNASIS (Hitachi System Engineering Co., Japan).

FIG. 6 shows a mouse pancreatic β-cell strain MIN6-derived Gprotein-coupled receptor protein-encoding DNA (SEQ ID NO:27) and anamino acid sequence (SEQ ID NO:22) encoded by the isolated DNA basedupon the nucleotide sequences of plasmids pG3-2 and pG1-10 which areheld by the transformant Escherichia coli INVαF′/pG3-2. The underlinedportions represent regions corresponding to the synthetic primers.

Homology retrieval was carried out based upon the determined necleotidesequence [FIG. 6]. As a result, it was learned that a novel Gprotein-coupled receptor protein was encoded by the cDNA fragmentobtained. To further confirm this fact, by using DNASIS (Hitachi SystemEngineering Co., Japan) the nucleotide sequence was converted into anamino acid sequence [FIG. 6], hydrophobicity plotting was carried out toconfirm the presence of six hydrophobic regions [FIG. 8]. Upon comparingthe amino acid sequence with that of p19P2 obtained in Example 2,furthermore, a high degree of homology was found as shown in [FIG. 7].As a result, it is strongly suggested that the G protein-coupledreceptor proteins encoded by pG3-2 and pG1-10 recognize the same ligandas the G protein-coupled receptor protein encoded by p19P² does whilethe animal species from which the receptor proteins encoded by pG3-2 andpG1-10 are derived is different from that from which the receptorprotein encoded by p19P2 is.

Example 5

Cloning of cDNA Comprising Whole Coding Regions for Receptor Proteinfrom Human Pituitary Gland-Derived cDNA Library

The DNA library constructed by Clontech Co. wherein λ gt11 phage vectoris used (CLONTECH Laboratories, Inc.; CLH L1139b) was employed as ahuman pituitary gland-derived cDNA library. The human pituitary glandcDNA library (2×10⁶ pfu (plaque forming units)) was mixed with E. coliY1090 treated with magnesium sulfate, and incubated at 37° C. for 15minutes followed by addition of 0.5% agarose (Pharmacia Co.) LB. The E.coli was plated onto a 1.5% agar (Wako-Junyaku Co.) LB plate (containing50 μg/ml of ampicillin). A nitrocellulose filter was placed on the plateon which plaques were formed and the plaque was transferred onto thefilter. The filter was denatured with an alkali and then heated at 80°C. for 3 hours to fix DNAs.

The filter was incubated overnight at 42° C. together with the probementioned herein below in a buffer containing 50% formamide, 5×SSPE(20×SSPE (pH 7.4) is 3 M NaCl, 0.2 M NaH₂PO₄H₂O, 25 mM EDTA), 5×Denhardt's solution (Nippon Gene, Japan), 0.1% SDS and 100 μg/ml ofsalmon sperm DNA for hybridization.

The probe used was obtained by cutting the DNA fragment inserted in theplasmid, p19P2, obtained in Working Example 2, with EcoRI, followed byrecovery and labelling by incorporation of [³²P]dCTP (Dupont Co.) with arandom prime DNA labelling kit (Amasham Co.).

It was washed with 2×SSC (20×SSC is 3 M NaCl, 0.3 M sodium citrate),0.1% SDS at 55° C. for 1 hour and, then, subjected to an autoradiographyat −80° C. to detect hybridized plaques.

In this screening, hybridization signals were recognized in threeindependent plaques. Each DNA was prepared from the three clones. TheDNAs digested with EcoRI were subjected to an agarose electrophoresisand were analyzed by the southern blotting using the same probe as theone used in the screening. Hybridizing bands were identified at about0.7 kb, 0.8 kb and 2.0 kb, respectively. Among them, the DNA fragmentcorresponding to the band at about 2.0 kb (λ hGR3) was selected. The λhGR3-derived EcoRI fragment with a hybridizable size was subcloned tothe EcoRI site of the plasmid, pUC18, and E. coli JM109 was transformedwith the plasmid to obtain transformant E. coli JM109/phGR3. Arestriction enzyme map of the plasmid, phGR3, was prepared relying upona restriction enzyme map deduced from the nucleotide sequence as shownin Example 2. As a result, it was learned that it carried a full-lengthreceptor protein-encoding DNA which was predicted from the receptorprotein-encoding DNA as shown in Example 2.

Example 6

Sequencing of Human Pituitary Gland-Derived Receptor Protein cDNA

Among the EcoRI fragments inserted in the plasmid, phGR3, obtained inthe above Example 5, the from EcoRI to NheI nucleotide sequence withabout 1330 bp that is considered to be a receptor protein-coding regionwas sequenced. Concretely speaking, by utilizing restriction enzymesites that exist in the EcoRI fragments, unnecessary parts were removedor necessary fragments were subcloned in order to prepare templateplasmids for analyzing the nucleotide sequence.

The reaction for determining the nucleotide sequence (sequencing) wascarried out with a DyeDeoxy terminator cycle sequencing kit (ABI Co.),the DNA was decoded with the fluorescent automatic sequencer (ABI Co.),and the data of the nucleotide sequence obtained were analyzed withDNASIS (Hitachi System Engineering Co., Japan).

FIG. 9 shows a nucleotide sequence of from immediate after the EcoRIsite up to the NheI site encoded by phGR3. The nucleotide sequence ofthe human pituitary gland-derived receptor protein-encoding DNAcorresponds to the nucleotide sequence (SEQ ID NO:26) of from 118th to1227th nucleotides (FIG. 9). An amino acid sequence of the receptorprotein that is encoded by the nucleotide sequence is shown in SEQ IDNO:21.

Example 7

Northern Hybridization with Human Pituitary Gland-Derived ReceptorProtein-Encoding phGR3

Northern blotting was carried out in order to detect the expression ofphGR3-encoded human pituitary gland-derived receptor proteins obtainedin Example 5 in the pituitary gland at a mRNA level. Human pituitarygland mRNA (2.5 μg, Clontech Co.) was used as a template mRNA and thesame as the probe used in Working Example 5 was used as a probe. Nylonmembrane (Pall Biodyne, U.S.A.) was used as a filter for northernblotting and migration of the mRNA and adsorption (sucking) thereof withthe blotting filter was carried out according to the method as disclosedin Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989.

The hybridization was effected by incubating the above-mentioned filterand probe in a buffer containing 50% formamide, 5×SSPE, 5× Denhardt'ssolution, 0.1% SDS and 100 μg/ml of salmon sperm DNA overnight at 42° C.The filter was washed with 0.1×SSC, 0.1% SDS at 50° C. and, after dryingwith an air, was exposed to an X-ray film (XAR5, Kodak) for three daysat −80° C. The results were as shown in FIG. 10 from which it isconsidered that the receptor gene encoded by phGR3 is expressed in thehuman pituitary gland.

Example 8

Amplification of Receptor cDNA by PCR Using MIN6-Derived cDNA andSequencing

By using, as a template, 5 μl of cDNA prepared from the mouse pancreaticβ-cell strain, MIN6 in Working Example 3, PCR amplification using theDNA primers synthesized in Example 4 as disclosed in Libert F. et al.,“Science, 244: 569-572, 1989”, i.e., a synthetic primer represented bythe following sequence: 5′-CTGTG (C or T) G (C or T) (SEQ ID NO: 31) (Gor C) AT (C or T) GCIIT (G or T) GA (C or T) (A or C) G (G or C) TAC-3′

-   wherein I is inosine; and

a synthetic primer represented by the following sequence: 5′-A (G or T)G (A or T) AG (A or (SEQ ID NO: 32) T) AGGGCAGCCAGCAGAI (G or C) (A orG) (C or T) GAA-3′

-   wherein I is inosine, was carried out under the same conditions as    in Example 1. The resulting PCR product was subcloned to the plasmid    vector, pCR™II, in the same manner as in Example 2 to obtain a    plasmid, p5S38. The plasmid p5S38 was transfected into E. coli JM109    to obtain transformant Escherichia coli JM109/p5S38.

The reaction for determining the nucleotide sequence (sequencing) wascarried out with a DyeDeoxy terminator cycle sequencing kit (ABI Co.),the DNA was decoded with the fluorescent automatic sequencer (ABI Co.),and the data of the nucleotide sequence obtained were read with DNASIS(Hitachi System Engineering Co., Japan).

FIG. 12 showns a mouse pancreatic β-cell strain MIN6-derived Gprotein-coupled receptor protein-encoding DNA (SEQ ID NO:28) and anamino acid sequence (SEW ID NO:23) encoded by the isolated DNA basedupon the nucleotide sequence of plasmid, p5S38. The underlined portionsrepresent regions corresponding to the synthetic primers.

Homology retrieval was carried out based upon the determined nucleotidesequence [FIG. 12]. As a result, it was learned that a novel Gprotein-coupled receptor protein was encoded by the cDNA fragmentobtained. To further confirm this fact, by using DNASIS (Hitachi SystemEngineering Co., Japan), the nucleotide sequence was converted into anamino acid sequence [FIG. 12], and hydrophobicity plotting was carriedout to confirm the presence of four hydrophobic regions [FIG. 14]. Uponcomparing the amino acid sequence with those encoded by p19P2 obtainedin Example 2 and encoded by pG3-2 obtained in Example 4, furthermore, ahigh degree of homology was found as shown in FIG. 13. As a result, itis strongly suggested that the mouse pancreatic β-cell strain,MIN6-derived G protein-coupled receptor protein encoded by p5S38recognizes the same ligand as the human pituitary gland-derived Gprotein-coupled receptor protein encoded by p19P2 does while the animalspecies from which the receptor protein encoded by p5S38 is derived isdifferent from that from which the receptor protein encoded by p19P2 is.It is also strongly suggested that the mouse pancreatic β-cell strain,MIN6-derived G protein-coupled receptor protein encoded by p5S38recognized the same ligand as the mouse pancreatic β-cell strain,MIN6-derived G protein-coupled receptor proteins encoded by pG3-2 andpG1-10 do and they are analogous receptor proteins one another(so-called “subtype”).

Example 9

Preparation of CHO Cells Which Express phGR3

The plasmid phGR3 (Example 5) containing a cDNA encoding the full-lengthamino acid sequence of human pituitary receptor protein was digestedwith the restriction enzyme Nco I and electrophoresed on agarose gel anda fragment of about 1 kb was recovered. Both ends of the recoveredfragment were blunted with a DNA blunting kit (Takara Shuzo Co., Japan)and, with the SalI linker added, treated with SalI and inserted into theSalI site of pUC119 to provide plasmid S10. Then, S10 was treated withSalI and SacII to prepare a fragment of about 700 bp (containing theN-terminal coding region). Then, a fragment of about 700 bp (containingthe C-terminal coding region including initiation and terminationcodons) was cut out from phGR3 with Sac II and Nhe I. These twofragments were added to the animal cell expression vector plasmidpAKKO-111H (the vector plasmid identical to the pAKKO1.11 H described inBiochim. Biophys. Acta, Hinuma, S., et al., 1219 251-259, 1994) and aligation reaction was carried out to construct a full-length receptorprotein expression plasmid pAKKO-19P2.

E. coli transfected with pAKKO-19P2 was cultured and the pAKKO-19P2plasmid DNA was mass-produced using QUIAGEN Maxi. A 20 μg portion of theplasmid DNA was dissolved in 1 ml of sterile PBS, and in a gene transfervial (Wako Pure Chemical Ind.), the solution was vortexed well forriposome formation. This riposome, 125 μl, was added to CHOdhfr⁻ cellssubcultured at 1×10⁶ per 10 cm-dia. dish 24 hr before and placed infresh medium immediately before addition and overnight culture wascarried out. After a further one-day culture in fresh medium, the mediumwas changed to a screening medium and the incubation was further carriedout for a day. For efficient screening of transformants, subculture wascarried out at a low cell density and only the cells growing in thescreening medium were selected to establish a full-length receptorprotein expression CHO cell line CHO-19P2.

Example 10

Confirmation of the Amount of Expression of the Full-Length ReceptorProtein in the CHO-19P2 Cell Line at the Transcription Level

Using FastTrack Kit (Invitrogen), CHO cells transfected with pAKKO-19P2according to the kit manual and mock CHO cells were used to preparepoly(A)⁺RNA. Using 0.02 μg of this poly(A)⁺RNA, a cDNA was synthesizedby means of RNA PCR Kit (Takara Shuzo, Co., Japan). The kind of primerused was a random 9mer and the total volume of the reaction mixture was40 μl. As a negative control of cDNA synthesis, a reversetranscriptase-free reaction mixture was also provided. First, thereaction mixture was incubated at 30° C. for 10 minutes to conduct anamplification reaction to some extent. Then, it was incubated at 42° C.for 30 minutes to let the reverse transcription reaction proceed. Theenzyme was inactivated by heating at 99° C. for 5 minutes and thereaction system was cooled at 5° C. for 5 minutes.

After completion of the reverse transcription reaction, a portion of thereaction mixture was recovered and after dilution with distilled water,extraction was carried out with phenol/chloroform and further withdiethyl ether. The extract was subjected to precipitation from ethanoland the precipitate was dissolved in a predetermined amount of distilledwater for use as a cDNA sample. This cDNA solution and the plasmid DNA(pAKKO-19P2) were serially diluted and using primers specific tofull-length receptor protein, PCR was carried out. The sequences of theprimers prepared according to the base sequence of the coding region ofthe full-length receptor protein were CTGACTTATTTTCTGGGCTGCCGC (SEQ IDNO:33) for 5′ end and AACACCGACACATAGACGGTGACC (SEQ ID NO:34) for 3′end.

The PCR reaction was carried out in a total volume of 100 μl using 1 μMeach of the primers, 0.5 μl of Taq DNA polymerase (Takara Shuzo Co.,Japan), the reaction buffer and dNTPs accompanying the enzyme, and 10 μlof template DNA (cDNA or plasmid solution). First the reaction mixturewas heat-treated at 94° C. for 2 minutes for sufficient denaturation ofthe template DNA and subjected to 25 cycles of 95° C.×30 seconds, 65°C.×30 seconds, and 72° C.×60 seconds. After completion of the reaction,10 μl of the reaction mixture was subjected to agarose gelelectrophoresis and the detection and quantitative comparison ofamplification products were carried out. As a result, a PCR product ofthe size (400 bp) predictable from the sequence of the cDNA coding forthe full-length receptor protein was detected [FIG. 15]. In the lane ofthe PCR reaction mixture using the product of the reversetranscriptase-free transcription system as the template, no specificband was detected, thus extruding the possibility of its being a PCRproduct derived from the genomic DNA of CHO cells. Moreover, no specificband appeared in the lane of mock cells, either. Therefore, it was clearthat the product was not derived from the mRNA initially expressed inCHO cells [FIG. 15].

Example 11

Detection of the Activity to Specifically Promote Release of ArachidonicAcid Metabolites from CHO-19P2 Cells in a Rat Whole Brain Extract

A crude peptide fraction was prepared from rat whole brain by thefollowing procedure. The rat whole brain enucleated immediately aftersacrifice was frozen in liquefied nitrogen and stored at −80° C. Thefrozen rat whole brain, 20 g (the equivalent of 10 rats) was finelydivided and boiled in 80 ml of distilled water for 10 minutes. After theboiled tissue was quenched on ice, 4.7 ml of acetic acid was added at afinal concentration of 1.0 M and the mixture was homogenized using aPolytron (20,000 rpm, 6 min.). The homogenate was stirred overnight andthen centrifuged (10,000 rpm, 20 min.) to separate the supernatant. Thesediment was homogenized in 40 ml of 1.0 M acetic acid and centrifugedagain to recover the supernatant. The supernatants were pooled, dilutedin 3 volumes of acetone, allowed to stand on ice for 30 minutes, andcentrifuged (10,000 rpm, 20 min.) to recover the supernatant. Therecovered supernatant was evaporated to remove acetone. To the resultingacetone-free concentrate was added 2 volumes of 0.05% trifluoroaceticacid (TFA)/H₂O and the mixture was applied to a reversed-phase C18column (Prep C18 125A, Millipore). After application of the supernatant,the column was washed with 0.05% TFA/H₂O, and gradient elution wascarried out with 10%, 20%, 30%, 40%, 50%, and 60% CH₃CN/0.05% TFA/H₂O.The fractions were respectively divided into 10 equal parts andlyophilized. The dried sample derived from one animal equivalent of ratwhole brain was dissolved in 20 μl of dimethyl sulfoxide (DMSO) andsuspended in 1 ml of Hank's balanced saline solution (HBSS) supplementedwith 0.05% bovine serum albumin (BSA) to provide a crude peptidefraction.

The full-length receptor protein-expressed CHO cells and mock CHO cellswere seeded in a 24-well plate, 0.5×10⁵ cells/well, and cultured for 24hours. Then, [³H] arachidonic acid was added at a final concentration of0.25 μCi/well. Sixteen (16) hours after addition of [³H] arachidonicacid, the cells were rinsed with 0.05% BSA-HBSS and the above-mentionedcrude peptide fraction was added, 400 μl/well. The mixture was incubatedat 37° C. for 30 minutes and a 300 μl portion of the reaction mixture(400 μl) was added to 4 ml of a scintillator and the amount of [³H]arachidonic acid metabolite released into the reaction mixture wasdetermined with a scintillation counter. As a result, an arachidonicacid metabolite-releasing activity specific to the full-length receptorprotein expressed CHO cells (CHO-19P2) was detected in the 30% CH₃CNfraction of the eluate [FIG. 16].

Example 12

Detection of the Activity to Specifically Promote Release of ArachidonicAcid Metabolites from CHO-19P2 Cells in a Bovine Hypothalamus Extract

A crude peptide fraction was prepared from 360 g (the equivalent of 1animals) of bovine brain tissue including hypothalamus in the samemanner as in Example 11. A dried peptide sample per 0.05 animal wasdissolved in 40 μl of DMSO and suspended in 2 ml of 0.05% BSA-HBSS andthe detection of arachidonic acid metabolite-releasing activity wasattempted in the same manner as in Example 11. As a result, the activityto specifically promote release of arachidonic acid metabolites from theCHO-19P2 cell line was detected in the fraction eluted with 30% CH₃CNfrom a C18 column to which the crude bovine hypothalamus peptidefraction had been applied [FIG. 17].

Example 13

Preparation of the Activity (Peptide) to Specifically Promote Release ofArachidonic Acid Metabolites from CHO-19P2 Cells by Purification fromBovine Hypothalamus

A typical process for harvesting the activity to specifically promoterelease of arachidonic acid metabolites from the CHO-19P2 cell line bypurification from bovine hypothalamus is now described. A frozen bovinebrain tissue specimen including hypothalamus, 4.0 kg (the equivalent of80 animals) was ground and boiled in 8.0 L of distilled water for 20minutes. After quenching on ice, 540 ml of acetic acid was added at afinal concentration of 1.0 M and the mixture was homogenized using aPolytron (10,000 rpm, 12 min.). The homogenate was stirred overnight andthen centrifuged (9,500 rpm, 20 min) to recover a supernatant. Thesediment was suspended in 4.0 L of 1.0 M acetic acid and homogenizedwith the Polytron and centrifuged again to recover a furthersupernatant. The supernatants were pooled and TFA was added at a finalconcentration of 0.05%. The mixture was applied to reversed-phase C18(Prep C18 125A % 160 ml; Millipore) packed in a glass column. Afteraddition, the column was washed with 320 ml of 0.05% TFA/H₂O and3-gradient elution was carried out with 10%, 30%, and 50% CH₃CN/0.05%TFA/H₂O. To the 30% CH₃CN/0.05% TFA/H₂O fraction was added 2 volumes of20 mM CH₃COONH₄/H₂O and the mixture was applied to the cation exchangecolumn HiPrep CM-Sepharose FF (Pharmacia). After the column was washedwith 20 mM CH₃COONH₄/10% CH₃CN/H₂O, 4-gradient elution was carried outwith 100 mM, 200 mM, 500 mM, and 1000 mM CH₃COONH₄/10% CH₃CN/H₂O. In the200 mM CH₃COONH₄ fraction, activity to specifically promote release ofarachidonic acid metabolites from CHO-19P2 was detected. Therefore, thisfraction was diluted with 3 volumes of acetone, centrifuged fordeproteination, and concentrated in an evaporator. To the concentratedfraction was added TFA (final concentration 0.1%) and the mixture wasadjusted to pH 4 with acetic acid and applied to 3 ml of thereversed-phase column RESOURCE RPC (Pharmacia). Elution was carried outon a concentration gradient of 15%-30% CH₃CN. As a result, activity tospecifically promote the release of arachidonic acid metabolites fromthe CHO-19P2 cell line was detected in the 19%-21% CH₃CN fraction. Theactive fraction eluted from RESOURCE RPC was lyophilized, dissolved withDMSO, suspended in 50 mM MES pH 5.0/10% CH₃CN, and added to 1 ml of thecation exchange column RESOURCE S. Elution was carried out on aconcentration gradient of 0 M-0.7 M NaCl. As a result, the activity tospecifically promote release of arachidonic acid metabolites fromCHO-19P2 cells was detected in the 0.32 M-0.46 M NaCl fraction. Theactive eluate from RESOURCE S was lyophilized, dissolved with DMSO,suspended in 0.1% TFA/H₂O, and added to reversed-phase column C18218TP5415 (Vydac), and elution was carried out on a concentrationgradient of 20%-30% CH₃CN. As a result, the activity to specificallypromote release of arachidonic acid metabolites from CHO-19P2 cells wasdetected in the three fractions 22.5%, 23%, and 23.5% CH₃CN (theseactive fractions are designated as P-1, P-2, and P-3) (FIG. 18). Of thethree active fractions, the 23.5% CH₃CN fraction (P-3) was lyophilized,dissolved with DMSO, suspended in 0.1% TFA/H₂O, and added to thereversed-phase column diphenyl 219TP5415 (Vydac), and elution wascarried out on a gradient of 22%-25% CH₃CN. As a result, the activity tospecifically promote release of arachidonic acid metabolites fromCHO-19P2 cells was converged by recovered in one elution peak obtainedwith 23% CH₃CN [FIG. 19]. The peak activity fraction from thereverse-phased column diphenyl 219TP5415 was lyophilized, dissolved withDMSO, suspended in 0.1% TFA/H₂O, and added to the reversed-phase columnμRPC C2/C18 SC 2.1/10 (Pharmacia), and elution was carried out on agradient of 22%-23.5% CH₃CN. As a result, the activity to specificallypromote release of arachidonic acid metabolites from CHO-19P2 cells wasdetected in the two peaks eluted with 23.0% and 23.2% CH₃CN [FIG. 20].

Example 14

Determination of the Amino Acid Sequence of the Peptide Having theActivity to Specifically Promote Release of Arachidonic Acid Metabolitesfrom CHO-19P2 Cells as Purified from Bovine Hypothalamus

The amino acid sequence of the peptide (P-3) having activity tospecifically promote release of arachidonic acid metabolites fromCHO-19P2 cells as purified in Example 13 was determined. The fraction ofpeak activity from the reversed-phase μRPC C2/C18 SC 2.1/10 waslyophilized and dissolved in 20 μl of 70% CH₃CN and analyzed for aminoacid sequence with the peptide sequencer (ABI.491). As a result, thesequence defined by SEQ ID NO:3 was obtained. However, the 7th and 19thamino acids were not determined by only the analysis of amino acidsequence.

Example 15

Preparation of the Active Substance (Peptide) Which SpecificallyPromotes Release of Arachidonic Acid Metabolites from CHO-19P2 Cells asPurified from Bovine Hypothalamus

Of the three active fractions obtained with Vydac C18 218TP5415 inExample 13, the active fraction (P-2) eluted with 23.0% CH₃CN wasfurther purified. This active fraction was lyophilized, dissolved withDMSO, suspended in 0.1% TFA/dH₂O, and added to reversed-phase columndiphenyl 219TP5415 (Vydac), and elution was carried out on a gradient of21.0%-24.0% CH₃CN. As a result, activity to specifically promote releaseof arachidonic acid metabolites from CHO-19P2 cells was detected in apeak eluted with 21.9% CH₃CN. This fraction was lyophilized, dissolvedwith DMSO, suspended in 0.1% TFA/dH₂O, and added to reversed-phase μRPCC2/C18 SC 2.1/10 (Pharmacia), and elution was carried out on a CH₃CNgradient of 21.5%-23.0%. As a result, the activity to specificallypromote release of arachidonic acid metabolites from CHO-19P2 cellsconverged in one peak eluted with 22.0% CH₃CN [FIG. 21].

Example 16

Determination of the Amino Acid Sequence of the Peptide (P-2) Purifiedfrom Bovine Hypothalamus Which Specifically Promotes Release ofArachidonic Acid Metabolites from CHO-19P2 Cells

The amino acid sequence of the peptide (P-2) having the activity tospecifically promote release of arachidonic acid metabolites fromCHO-19P2 cells as purified in Example 15 was determined. The peakactivity fraction from the reversed-phase column μRPC C2/C18 SC 2.1/10was lyophilized, dissolved in 20 μl of 70% CH₃CN, and analyzed for aminoacid sequence with the peptide sequencer (ABI, 492) (SEQ ID NO:4).

Example 17

Preparation of a Poly(A)⁺RNA Fraction from Bovine Hypothalamus andSynthesis of a cDNA

Using Isogen (Nippon Gene), total RNA was prepared from one animalequivalent of bovine hypothalamus. Then, using Fast Track (Invitrogen),a poly(A)⁺RNA fraction was prepared. From 1 μg of this poly(A)⁺RNAfraction, cDNA was synthesized using 3′ RACE system (GIBCO BRL) andMarathon cDNA amplification kit (Clontech) according to the manuals anddissolved in 20 and 10 μl, respectively.

Example 18

Acquisition of cDNA Coding for the amino acid Sequence established inExample 14

To obtain a cDNA coding for a polypeptide comprising the amino acidsequence established in Example 14, the acquisition of a base sequencecoding for SEQ ID NO:1 was attempted in the first place. Thus, primersP5-1 (SEQ ID NO:35), P3-1 (SEQ ID NO:36), and P3-2 (SEQ ID NO:37) weresynthesized. (In the Sequence Table, I represents inosine). Using 0.5 μlof the cDNA prepared by 3′ RACE in Example 17 as a template and EXTaq(Takara Shuzo Co., Japan) as DNA polymerase, 2.5 μl of accompanyingbuffer, 200 μM of accompanying dNTP, and primers P5-1 and P3-1 wereadded each at a final concentration of 200 nM, with water added to make25 μl, and after one minute at 94° C., the cycle of 98° C.×10 seconds,50° C.×30 seconds, 68° C.×10 seconds was repeated 30 times. Thisreaction mixture was diluted 50-fold with tricine-EDTA buffer and using2.5 μl of the dilution as a template and the primer combination of P5-1and P3-2, the reaction was carried out in otherwise the same manner asdescribed above. As the thermal cycler, Gene Amp 9600 (Perkin Elmer) wasused. The amplification product was subjected to 4% agaroseelectrophoresis and ethidium bromide staining and a band of about 70 bpwas cut out and subjected to thermal fusion, phenol extraction, andethanol precipitation. The recovered DNA was subcloned into plasmidvector PCR™II according to the manual of TA Cloning kit (Invitrogen).The vector was then introduced into E. coli JM109 and the resultanttransformant was cultured in ampicillin-containing LB medium. Theplasmid obtained with an automatic plasmid extractor (Kurabo) wasreacted according to the manual of Dye Terminator Cycle Sequencing Kit(ABI) and decoded with a fluorescent automatic DNA sequencer (ABI). As aresult, the sequence shown in FIG. 22 was obtained and confirmed to bepart of the base sequence coding for SEQ ID NO:1.

Example 19

Acquisition of a Bioactive Polypeptide cDNA by RACE Using the SequenceEstablished in Example 18

First, for amplification (5′ RACE) of the sequence at 5′ end, the twoprimers PE (SEQ ID NO:38) and PDN (SEQ ID NO:39) were synthesized byutilizing the sequence shown in FIG. 22. The cDNA prepared usingMarathon cDNA amplification kit in Example 17 was diluted 100-fold withtricine-EDTA buffer. Then, in the same manner as Example 2, a reactionmixture was prepared using 2.5 μl of the dilution and a combination ofthe adapter primer AP1 accompanying the kit and the primer PE and afterone minute at 94° C., the cycle of 98° C.×10 seconds and 68° C.×5minutes was repeated 30 times. This reaction system was further diluted50-fold with tricine-EDTA buffer and using 2.5 μl of the dilution as atemplate and the changed primer combination of AP1 and PDN, the reactionwas conducted at 94° C. for one minute, followed by 4 cycles of 94° C.×1minute, 98° C.×10 seconds, 72° C.×5 minutes, 4 cycles of 98° C.×10seconds, 70° C.×5 minutes, and 26 cycles of 98° C.×10 seconds, 68° C.×5minutes. The amplification product was electrophoresed on 1.2% agarosegel and stained with ethidium bromide and a band of about 150 bp was cutout and centrifugally filtered through a centrifugal filter tube(Millipore), extracted with phenol, and precipitated from ethanol. Therecovered DNA was subcloned into plasmid vector PCR™II according to themanual of TA Cloning Kit (Invitrogen). The vector was then introducedinto E. coli JM109 and the resulting transformant was cultured and thesequence of the inserted cDNA fragment was analyzed as in Example 18. Asa result, the sequence shown in FIG. 23 was obtained. Based on thissequence, primers FB (SEQ ID NO:40) and FG (SEQ ID NO:41) weresynthesized and the 3′ sequence was cloned (3′ RACE). Using the sametemplate as that for 5′ RACE in the same quantity and the combination ofthe accompanying adapter primer AP1 with the primer FC, PCR was carriedout at 94° C. for 1 minute, followed by 5 cycles of 98° C.×10 seconds,72° C.×5 minutes, 5 cycles of 98° C.×10 seconds, 70° C.×5 minutes, and25 cycles of 98° C.×10 seconds, 68° C.×5 minutes. Then, using 2.5 μl ofa 50-fold dilution of this reaction mixture in tricine-EDTA buffer asthe template and the combination of the accompanying primer AP2 with theprimer FB, the reaction was further conducted at 94° C. for one minute,followed by 4 cycles of 98° C.×10 seconds, 72° C.×5 minutes, 4 cycles of98° C.×10 seconds, 70° C.×5 minutes, and 27 cycles of 98° C.×10 seconds,68° C.×5 minutes. The amplification product was electrophoresed on 1.2%agarose gel and stained with ethidium bromide and a band of about 400 bpwas cut out and the DNA was recovered as in 5′-RACE. This DNA fragmentwas subcloned into plasmid vector pCR™II and introduced into E. coliJM109 and the sequence of the inserted cDNA fragment in the resultingtransformant was analyzed. From the results of 5′ RACE and 3′ RACE, theDNA sequence [FIG. 24] coding for the complete coding region of thebioactive polypeptide defined by SEQ ID NO:1 was established. Thus, inFIGS. 24(a) and (b), the base¹³⁴ is G, the base¹⁸⁴ is T or C, and thebase²⁴⁵ was T or C.

The cDNA shown in FIG. 24 was the cDNA encoding a polypeptide consistingof 98 amino acids. The fact that the amino acids in 1-22-positionscomprise a cluster of hydrophobic amino acids taken together with thefact that the N-terminal region of the active peptide begins with Ser in23-position as shown in Example 14 suggested that the amino acids 1-22represent a secretion signal sequence. On the other hand, the Gly ArgArg Arg sequence in 54-57 positions of the polypeptide was found to be atypical amino acid sequence motif which exists in the event of cleavageof a bioactive peptide. As it is the case with this cleavage motif, itis known that because of the presence of Gly, the C-terminus of theproduct peptide is frequently amidated.

The P-3 N-terminal sequence data of Example 14 and P-2 N-terminalsequence data in Example 16 coupled with this GlyArgArgArg sequencesuggest that at least same of the bioactive peptides cut out from thepolypeptide encoded by this cDNA are defined by SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 orSEQ ID NO:10.

Example 20

Acquisition of a DNA Fragment Comprising the Full Coding Region ofBovine-Derived Bioactive Polypeptide cDNA by PCR

Using the cDNA prepared with Marathon cDNA amplification kit in Example17 as a template, a DNA fragment including the entire coding region ofbioactive polypeptide cDNA was constructed. First, based on the sequenceof cDNA elucidated in Example 19, two primers having base sequencesdefined by SEQ ID NO:42 and SEQ ID NO:43, respectively, weresynthesized. BOVF 5′-GTGTCGACGAATGAAGGCGGTGGGGGC (SEQ ID NO: 42)CTGGC-3′ BOVR (24 mer) 5′-AGGCTCCCGCTGTTATTCCTGGAC-3′ (SEQ ID NO: 43)

BOVF contains the initiation codon of bioactive polypeptide cDNA and isa sense sequence corresponding to −2-+22 (A of the initiation codon ATGbeing reckoned as +1) with restriction enzyme SalI site added. On theother hand, BOVR is an antisense sequence corresponding to +285-+309which includes the termination codon of bioactive polypeptide cDNA.

The PCR was conduced as follows. The cDNA prepared using Marathon cDNAamplification kit in Example 17 was diluted 100-fold in tricine-EDTAbuffer and using 2.5 μl of the dilution, a reaction mixture was preparedas in Example 2 and subjected to 94° C.×1 minute, 3 cycles of 98° C.×10seconds, 72° C.×5 minutes, 3 cycles of 98° C.×10 seconds, 70° C.×5minutes, and 27 cycles of 98° C.×10 seconds, 68° C.×5 minutes. Theamplification product was subjected to 2% agarose electrophoresis andethidium bromide staining and a band of about 320 bp was cut out. TheDNA was recovered and subcloned in plasmid vector PCR™II as in Example3. The vector was introduced into Escherichia coli JM109 to provide thetransformant E. coli JM109/pBOV3. The sequence of the cDNA fragmentinserted in the transformant was then analyzed. As a result, this DNAfragment was confirmed to be a fragment covering the entire codingregion of the bioactive polypeptide cDNA.

Example 21

Synthesis ofSer-Arg-Ala-His-Gln-His-Ser-Met-Glu-Ile-Arg-Thr-Pro-Asp-Ile-Asn-Pro-Ala-Trp-Tyr-Ala-Gly-Arg-Gly-Ile-Arg-Pro-Val-Gly-Arg-Phe-NH2(19P2-L31)

1) Synthesis ofSer(Bzl)-Arg(Tos)-Ala-His(Bom)-Gln-His(Bom)-Ser(Bzl)-Met-Glu(OcHex)-Ile-Arg(Tos)-Thr(Bzl)-Pro-Asp(OcHex)-Ile-Asn-Pro-Ala-Trp(CHO)-Tyr(Br-Z)-Ala-Gly-Arg(Tos)-Gly-Ile-Arg(Tos)-Pro-Val-Gly-Arg(Tos)-Phe-pMBHA-resin

The reactor of a peptide synthesizer (Applied Biosystems 430A) wascharged with 0.71 g (0.5 mmole) of commercial p-methyl-BHA resin(Applied Biosystems, currently Perkin Elmer). After wetting with DCM,the initial amino acid Boc-Phe was activated by the HOBt/DCC method andintroduced into the p-methyl-BHA resin. The resin was treated with 50%TFA/DCM to remove Boc and make the amino group free and neutralized withDIEA. To this amino group was condensed the next amino acid Boc-Arg(Tos) by the HOBt/DCC method. After the absence of unreacted aminofunction was verified by ninhydrin test, a sequential condensation ofBoc-Gly, Boc-Val, Boc-Pro, Boc-Arg(Tos), Boc-Ile, Boc-Gly, Boc-Arg(Tos),Boc-Gly, Boc-Ala, Boc-Tyr(Br-Z) was carried out. The Boc-Ala, Boc-Tyr(Br-Z), the condensation of which was found insufficient by ninhydrintest, was recondensed to complete the reaction. The resin was dried anda half of the resin was withdrawn. To the remainder, Boc-Trp(CHO),Boc-Ala, Boc-Pro, Boc-Asn, Boc-Ile, Boc-Asp(OcHex), Boc-Pro,Boc-Thr(Bzl), Boc-Arg(Tos), Boc-Ile, Boc-Glu(OcHex), Boc-Met,Boc-Ser(Bzl), Boc-His(Bom), Boc-Gln, Boc-His(Bom), Boc-Ala,Boc-Arg(Tos), Boc-Ser(Bzl) were serially condensed and recondensed untilsufficient condensation was confirmed by ninhydrin test. Afterintroduction of the full sequence of amino acids of 19P2-L31, the resinwas treated with 50% TFA/DCM to remove Boc groups on the resin and,then, dried to provide 1.28 g of the peptide resin.

2) Synthesis ofSer-Arg-Ala-His-Gln-His-Ser-Met-Glu-Ile-Arg-Thr-Pro-Asp-Ile-Asn-Pro-Ala-Trp-Tyr-Ala-Gly-Arg-Gly-Ile-Arg-Pro-Val-Gly-Arg-Phe-NH2(19P2-L31)

In a Teflon hydrogen fluoride reactor, the resin obtained in 1) wasreacted with 3.8 g of p-cresol, 1 ml of 1,4-butanedithiol, and 10 ml ofhydrogen fluoride at 0° C. for 60 minutes. The hydrogen fluoride and1,4-butanedithiol (1 ml) were distilled off under reduced pressure andthe residue was diluted with 100 ml of diethyl ether, stirred, filteredth rough a glass filter, and the fraction on the filter was dried. Thisfraction was suspended in 50 ml of 50% acetic acid/H₂O and stirred toextract the peptide. After separation of the resin, the extract wasconcentrated under reduced pressure to about 5 ml and chromatographed onSephadex G-25 (2×90 cm). Development was carried out with 50% aceticacid/H₂O and the 114 ml-181 ml fraction was pooled and lyophilized torecover 290 mg of white powders containing 19P2-L31. The powders wereapplied to a reversed-phase column of LiChroprep RP-18 (Merck) andrepeatedly purified by gradient elution using 0.1% TFA/H₂O and 0.1%TFA-containing 30% acetonitrile/H₂O. The fraction eluted at about 25%acetonitrile was pooled and lyophilized to provide 71 mg of whitepowders.

Mass spectrum (M+H)⁺ 3574.645

HPLC elution time 18.2 min.

Column conditions

-   -   Column: Wakosil 5C18 (4.6×100 mm)    -   Eluent:        -   A (0.1% TFA/H₂O)        -   B (0.1% TFA-containing 50 (% acetonitrile/H₂O)

Linear gradient elution from A to B (25 min.) Flow rate: 1.0 ml/min.

Example 22

Synthesis ofSer-Arg-Ala-His-Gln-His-Ser-Met(O)-Glu-Ile-Arg-Thr-Pro-Asp-Ile-Asn-Pro-Ala-Trp-Tyr-Ala-Gly-Arg-Gly-Ile-Arg-Pro-Val-Gly-Arg-Phe-NH2(19P2-L31(O))

In 20 ml of 5% acetic acid/H₂O was dissolved 6 mg of synthetic 19P2-L31and the Met only was selectively oxidized with 40 μl of 30% H₂O₂. Aftercompletion of the reaction, the reaction mixture was immediately appliedto a reversed-phase column of LiChroprep RP-18 (Merck) for purificationto provide 5.8 mg of the objective peptide.

Mass spectrum (M+H)⁺ 3590.531

HPLC elution time 17.9 min.

Column conditions

-   -   Column: Wakosil 5C18 (4.6×100 mm)    -   Eluent:        -   A (0.1% TFA/H₂O)        -   B (0.1% TFA-containing 50% aceto nitrile/H₂O)

Linear gradient elution from A to B (25 min.) Flow rate: 1.0 ml/min.

Example 23

Synthesis ofThr-Pro-Asp-Ile-Asn-Pro-Ala-Trp-Tyr-Ala-Gly-Arg-Gly-Ile-Arg-Pro-Val-Gly-Arg-Phe-NH2(19P2-L20)

To the resin subjected to condensations up to Boc-Tyr(Br-Z) in Example21-1) was further condensed Boc-Trp(CHO), Boc-Ala, Boc-Pro, Boc-Asn,Boc-Ile, Boc-Asp(OcHex), Boc-Pro, Boc-Thr(Bzl) serially in the samemanner to provide 1.14 g ofBoc-Thr(Bzl)-Pro-Asp(OcHex)-Ile-Asn-Pro-Ala-Trp(CHO)-Tyr(Br-Z)-Ala-Gly-Arg(Tos)-Gly-Ile-Arg(Tos)-Pro-Val-Gly-Arg(Tos)-Phe-pMBHA-resin.This resin was treated with hydrogen fluoride and columnwise purified inthe same manner as Example 21-2) to provide 60 mg of white powders.

Mass spectrum (M+H)⁺ 2242.149

HPLC elution time 10.4 min.

Column conditions

-   -   Column: Wakosil 5C18 (4.6×100 mm)    -   Eluent:        -   A (0.1% TFA-containing 15% aceto nitrile/H₂O)        -   B (0.1% TFA-containing 45% aceto nitrile/H₂O)

Linear gradient elution from A to B (15 min.) Flow rate: 1.0 ml/min.

Example 24

Determination of Arachidonic Acid Metabolites-Releasing Activity ofSynthetic Peptide (19P2-L31)

The activity of the peptide (19P2-L31) synthesized in Example 21 tospecifically release arachidonic acid metabolites from CHO-19P2 cellswas assayed in the same manner as Example 11. The synthetic peptide wasdissolved in degassed dH₂O at a concentration of 10⁻³M and diluted with0.05% BSA-HBSS and the activity to promote release of arachidonic acidmetabolites from CHO-19P2 cells at each concentration was assayed usingthe amount of [³H]arachidonic acid metabolites as the indicator. As aresult, concentration-dependent arachidonic acid metabolite-releasingactivity was detected over the range of 10⁻¹²M-10⁻⁶M [FIG. 25]. When thearachidonic acid metabolite-releasing activity of peptide 19P2-L31(O),i.e. the methionine-oxidation product of 19P2-L31 synthesized in Example22, was compared with that of 19P2-L31, it was found that the activityof 19P2-L31(O) was equivalent to the activity of 19P2-L31 as can be seenfrom FIG. 26.

Example 25

Determination of Arachidonic Acid Metabolites-Releasing Activity ofSynthetic Peptide (19P2-L20)

The activity of the synthetic equivalent (19P2-L20) of natural peptideP-2 as synthesized in Example 23 to specifically promote release ofarachidonic acid metabolites from CHO-19P2 cells was determined as inExample 11. Thus, the synthetic peptide was dissolved in degassed dH₂Oat a final concentration of 10⁻³M and this solution was serially dilutedwith 0.05% BAS-HBSS. The activity to specifically promote release ofarachidonic acid metabolites from CHO-19P2 cells at each concentrationwas assayed using the amount of [³H]arachidonic acid metabolites as theindicator.

As a result, concentration-dependent arachidonic acidmetabolite-releasing activity was detected over the range of 10⁻¹²-10⁻⁶M in nearly the same degree as 19P2-L31 [FIG. 27].

Example 26

Analysis of the Coding Region Base Sequence of Bovine Genomic DNA

pBOV3 was digested with restirction enzyme EcoRI and after fractionationby agarose gel electrophoresis, the DNA corressponding to the cDNAfragment was recovered to prepare a probe. This DNA was labeled with ³²Pusing a multiprime DNA labeling kit (Amersham). About 2.0×10⁶ phages ofBovine Genomic Library (Clontech BL1015j) constructed using cloningvector EMBL3 SP6/T7 and Escherichia coli K802 as the host were seeded inan LB agar plate and cultured overnight for plaque formation. Theplaques were transferred to a nitrocellulose filter and after alkalinemodification and neutralization, heat-treated (80° C., 2 hours) toinactivate the DNA. This filter was incubated with the labeled probe in50% formamide-Hybri buffer (50% formamide, 5× Denhardt solution, 4×SSPE,0.1 mg/ml heat-denatured salmon sperm DNA, 0.1% SDS) at 42° C. overnightfor hybridization. After this hybridization, the filter was washed with2×SSC, 0.1% SDS at room temperature for 1.5 hours, and further washed inthe same buffer at 55° C. for 30 minutes. Detection of the clonehybridizing with the probe was carried out on Kodak X-ray film(X-OMAT™AR) after 4 days of exposure using a sensitization screen at−80° C. After development of the film, the film was collated with platepositions and the phages which had hybridized were recovered. Then,plating and hybridization were repeated in the same manner for cloningof the pharges.

The cloned phages were prepared on a large scale by the plate lysatemethod and the phage DNA was extracted. Then, cleavage at therestriction enzyme SalI and BamHI cleavage sites at both ends of thecloning site of the vector and detection of the inserted fragmentderived from bovine genomic DNA was carried out by 1.2% agarose gelelectrophoresis [FIG. 28]. As a result, in the case of BamHI digestion,3 fragments were detected in addition to the bands derived from thephages. In the case of SalI digestion, one band overlapping the phageband was detected. The SalI-digested fragment being considered to harborthe full length and in order to subclone this fragment into a plasmidvector, it was ligated to BAP (E. coli-derived alkalinephosphatase)-treated plasmid vector pUC18 (Pharmacia) and introducedinto E. coli JM109. From this microorganism, a genome-derived SalIfragment-inserted plasmid DNA was prepared on a production scale and thebase sequence in the neighborhood of its coding region was analyzedusing Perkin Elmer Applied Biosystems 370A fluorecent sequencer and thesame manufacturer's kit. As a result, the sequence shown in FIG. 29 wasobtained. Comparison with the coding region of cDNA reveals that becauseof its being derived from genomic DNA, the coding region is divided intwo by a 472 bp intron [FIG. 30]. FIG. 31 and SEQ ID NO:44 present theamino acid sequence predicted from this bovine genome coding region(excluding the intron region).

Example 27

Preparation of Rat Medulla Oblongata Poly(A⁺)RNA Fraction and Synthesisof cDNA

Using Isogen (Nippon Gene), total RNA was prepared from the dorsalregion of rat medulla oblongata and using FastTrack (Invitrogen),poly(A)⁺RNA fraction was prepared. To 5 μg of this poly(A)⁺RNA was addedthe primer random DNA hexamer (BRL) and using Moloney mouse leukemiareverse transcriptase (BRL) and the accompanying buffer, complementaryDNA was synthesized. The reaction product was precipitated from ethanoland dissolved in 12 μl of DW. In addition, from 1 μg of thispoly(A)⁺RNA, a cDNA was synthesized using Marathon cDNA amplificationkit (Clontech) according to the manual and dissolved in 10 μl of DW.

Example 28

Acquisition of Rat Bioactive Polypeptide cDNA by RACE

To obtain the full coding region of rat bioactive polypeptide cDNA, anexperiment was performed in the same manner as the acquisition of bovinecDNA. First, PCR was carried out using the same primers P5-1 (SEQ IDNO:35) and P3-1 (SEQ ID NO:36) as used in Example 18 as primers and thecomplementary DNA synthesized in Example 27 using the primer random DNAhexamer (BRL) and Moloney mouse leukemia reverse transcriptase (BRL) asa template. The reaction system was composed of 1.25 μl of the templatecDNA, 200 μM of dNTP, 1 μM each of the primers, ExTaq (Takara Shuzo Co.,Japan) as DNA polymerase, and 2.5 μl of the accompanying buffer, with asufficient amount of water to make a total of 25 μl. The reaction wascarried out at 94° C. for 1 minute, followed by 40 cycles of 98° C.×10seconds, 50° C.×30 seconds, and 72° C.×5 seconds, and the reactionmixture was then allowed to stand at 72° C. for 20 seconds. The thermalcycler used was GeneAmp2400 (Perkin Elmer). The amplification productwas subjected to 4% agarose electrophoresis and ethidium bromidestaining and the band of about 80 bp was cut out. Then, in the mannerdescribed in Example 19, the DNA was recovered, subcloned into plasmidvector pCR™II, and introduced into E. coli JM109, and the inserted cDNAfragment was sequenced. As a result, a partial sequence of rat bioactivepolypeptide could be obtained. Based on this sequence, two primers,namely RA (SEQ ID NO:75) for 3′ RACE and RC (SEQ ID NO:76) for 5′ RACEwere synthesized and 5′ and 3′ RACEs were carried out. RA:5′-CARCAYTCCATGGAGACAAGAACCCC-3′ (SEQ ID NO: 75) (where R means A or G;Y means T or G) RC: 5′-TACCAGGCAGGATTGATACAGGGG-3′ (SEQ ID NO: 76)

As a template, the template synthesized using Marathon cDNAamplification kit (Clontech) in Example 27 was diluted 40-fold with theaccompanying tricine-EDTA buffer and 2.5 μl of the dilution was used. Asprimers, RA and the adapter primer AP1 accompanying the kit were usedfor 3′ RACE, and RC and AP1 for 5′ RACE. The reaction mixture wasprepared in otherwise the same manner as above. The reaction conditionswere 94° C.×1 minute, 5 cycles of 98° C.×10 seconds, 72° C.×45 seconds,3 cycles of 98° C.×10 seconds, 70° C.×45 seconds, and 40 cycles of 98°C.×10 seconds, 68° C.×45 seconds. As a result, a band of about 400 bpwas obtained from 3′ RACE and bands of about 400 bp and 250 bp from 5′RACE. These bands were recovered in the same manner as above and usingthem as templates and the primers used in the reaction, sequencing wascarried out with Dye Terminator Cycle Sequencing Kit (ABI). As a result,the sequence up to poly A could be obtained from the region consideredto be the 5′ noncoding region.

Example 29

Acquisition of the Full-Length cDNA of Rat Bioactive Polypeptide by PCR

Based on the sequence obtained in Example 28, two primers, viz. rF forthe region including the initiation codon (SEQ ID NO:77) and rR for the3′ side from the termination codon (SEQ ID NO:78), were synthesized toamplify the fragment including the full-length cDNA. rF:5′-GGCATCATCCAGGAAGACGGAGCAT-3′ (SEQ ID NO: 77) rR:5′-AGCAGAGGAGAGGGAGGGTAGAGGA-3′ (SEQ ID NO: 78)

Using the cDNA prepared using Moloney mouse leukemia reversetranscriptase in Example 27 as a template and ExTaq (Takara Shuzo Co.,Japan), PCR was carried out by repeating 40 cycles of 95° C.×30 seconds,68° C.×60 seconds. The amplification product was subjected to agaroseelectrophoresis and ethidium bromide staining and a band of about 350 bpwas cut out. The DNA was recovered, subcloned into plasmid vectorpCR™II, and introduced into E. coli JM109 as in Example 19. The plasmidwas extracted from the transformant and the base sequence wasdetermined. As a result, E. coli JM 109/pRAV3 having the full-lengthcDNA of rat bioactive polypeptide was obtained [FIG. 32].

Example 30

Synthesis of cDNA from the Human Total Brain Ply(A)⁺RNA Fraction

From 1 μg of human total brain poly(A)⁺RNA fraction (Clontech), cDNA wassynthesized with Marathon cDNA amplification kit (Clontech) according tothe manual and dissolved in 10 μl. In addition, the random DNA hexamer(BRL) was added as primer to 5 μg of the same poly(A)⁺RNA fraction andusing Moloney mouse leukemia reverse transcriptase (BRL) and theaccompanying buffer, complementary DNA was synthesized. The reactionproduct was precipitated from ethanol and dissolved in 30 μl of TE.

Example 31

Acquisition of Human Bioactive Polypeptide cDNA by RACE

From the amino acid sequence of rat bioactive polypeptide established inExample 28 [FIG. 33], the well-preserved regions of rat and bovinepolypeptides were selected and the following 3 primers R1 (SEQ IDNO:79), R3 (SEQ ID NO:80), and R4 (SEQ ID NO:81) were synthesized. Then,amplification of the region flanked by them was attempted by PCR usinghuman cDNA as a template. Referring to FIG. 33, bovine. aa representsthe amino acid sequence of bovine polypeptide, bovine. seq representsthe base sequence of the DNA coding for bovine polypeptide, and rat. seqrepresents the base sequence of the DNA coding for rat polypeptide. R1:5′-ACGTGGCTTCTGTGCTTGCTGC-3′ (SEQ ID NO: 79) R3:5′-GCCTGATCCCGCGGCCCGTGTACCA-3′ (SEQ ID NO: 80) R4:5′-TTGCCCTTCTCCTGCCGAAGCGGCCC-3′ (SEQ ID NO: 81)

The cDNA prepared using Marathon cDNA amplification kit (Clontech) inExample 30 was diluted 30-fold with tricine-EDTA buffer and 0.25 μl ofthe dilution was used as a template. The reaction mixture was composedof 200 μM of dNTP, 0.2 μM each of the primers R1 and R4, a 50:50 mixtureof Taq Start Antibody (Clontech) and DNA polymerase ExTaq (Takara ShuzoCo., Japan), 2.5 μl of the accompanying buffer, and a sufficient amountof water to make a total of 25 μl. The reaction conditions were 94° C.×1minute, followed by 42 cycles of 98° C.×10 seconds, 68° C.×40 seconds,and 1 minute of standing at 72° C. Then, using 1 μl of a 100-folddilution of the above reaction mixture in tricine-EDTA buffer as atemplate, the same reaction mixture as above except that the primercombination was changed to R1 and R3 was prepared and PCR was carriedout in the sequence of 94° C.×1 minute and 25 cycles of 98° C.×10seconds, 68° C.×40 seconds. The amplification product was subjected to4% agarose electrophoresis and ethidium bromide staining. As a result, aband of about 130 bp was obtained as expected. This band was recoveredin the same manner as in Example 28 and using the recovered fragment asa template, sequencing was carried out with Dye Terminator CycleSequencing Kit (ABI). As a result, a partial sequence of human bioactivepolypeptide could be obtained. Therefore, based on this sequence,primers HA (SEQ ID NO:82) and HB (SEQ ID NO:83) were synthesized for 3′RACE and primers HE (SEQ ID NO:84) and HF (SEQ ID NO:85) for 5′ RACE and5′ and 3′ RACEs were carried out. HA: 5′-GGCGGGGGCTGCAAGTCGTACCCATCG-3′(SEQ ID NO: 82) HB: 5′-CGGCACTCCATGGAGATCCGCACCCCT-3′ (SEQ ID NO: 83)HE: 5′-CAGGCAGGATTGATGTCAGGGGTGCGG-3′ (SEQ ID NO: 84) HF:5′-CATGGAGTGCCGATGGGTACGACTTGC-3′ (SEQ ID NO: 85)

As the template, 2.5 μl of a 20-fold dilution of the cDNA prepared inExample 30 in tricine-EDTA buffer was used. For the initial PCR,reaction mixtures were prepared in the same manner as above except thatHA and adapter primer AP1 were used for 3′ RACE and HE and AP1 for 5′RACE. The reaction sequence was 94° C.×1 minute, 5 cycles of 98° C.×10seconds, 72° C. for 35 seconds, 5 cycles of 98° C.×10 seconds, 70° C.×35seconds, and 40 cycles of 98° C.×10 seconds, 68° C.×35 seconds. Then,using 1 μl of a 100-fold dilution of this reaction mixture intricine-EDTA buffer as a template, a second PCR was carried out in thesame cycles as the first PCR. However, the reaction mixture was preparedusing primers HB and AP1 for 3′ RACE or HF and AP2 for 5′ RACE and KlenTaq (Clontech) as DNA polymerase and the accompanying buffer. As aresult, a band of about 250 bp was obtained from 3′ RACE and a band ofabout 150 bp from 5′-RACE. These bands were sequenced by the sameprocedure as above and using them in combination with the partialsequence obtained previously, the sequence from the region presumed tobe 5′-noncoding region to polyA of human bioactive polypeptide wasobtained.

Example 32

Acquisition of Human Bioactive Polypeptide Full-Length cDNA by PCR

Based on the sequence obtained in Example 31, two primers 5H (SEQ IDNO:86) and 3HN (SEQ ID NO:87) were synthesized for amplification of afragment including full-length cDNA. 5H:5′-GGCCTCCTCGGAGGAGCCAAGGGATGA-3′ (SEQ ID NO: 86) 3HN:5′-GGGAAAGGAGCCCGAAGGAGAGGAGAG-3′ (SEQ ID NO: 87)

Using 2.5 μl of the cDNA prepared using Moloney mouse leukemia reversetranscriptase (BRL) in Example 30 as a template and the reaction mixtureprepared using Klen Taq DNA polymerase (Clontech), the PCR reaction wasconducted in the sequence of 94° C.×1 minute and 40 cycles of 98° C.×10seconds, 68° C.×30 seconds. The fragment of about 360 bp obtained wasrecovered and subcloned (pCR™ 2.1 was used as the vector) in otherwisethe same manner as Example 29. The plasmid was recovered and its basesequence was determined. As a result, E. coli JM109/pHOV7 harboring thehuman bioactive polypeptide full-length cDNA was obtained [FIG. 34]. Inregard to the amino acid sequence of the translation region, acomparison was made between this human bioactive polypeptide and thebovine polypeptide shown in Example 20 or the rat polypeptide in Example29 [FIG. 35].

Example 33

An orphan G-protein coupled receptor, UHR-1, has been cloned from rathypothalamic suprachiasmic nuclei, and its nucleotide sequences havebeen reported (Biochemical and Biophysical Research Communications, vol.209, No. 2, pp 606-613, 1995, Genbank Accession Number: S77867). Aprotein coded by UHR-1 showed 91.6% identity over 359 amino acids withthat of phGR3, suggesting UHR-1 is a counterpart of hGR3. To confirmthis we cloned a cDNA for UHR-1 coding regions and established a CHOcells stably expressing UHR-1 as described below. Poly(A)⁺ RNA wasprepared from rat anterior pituitary using a FastTrack™ Kit (InvitrogenCo.), and cDNA was synthesized from 0.2 μg of this with Takara RNA PCRKit (Takara). The cDNA was dissolved in 10 μl of distilled water, andused as a template for the following PCR. To isolate UHR-1 cDNA, twoprimers, namely 5′-GTTCACAG(GTCGAC)ATGACCTCAC-3′ [SEQ ID NO:95] (UHF),and 5′-CTCAGA(GCTAGC)AGAGTGTCATCAG-3′ [SEQ ID NO:96] (UHR), weresynthesized on the basis of the sequence of UHR-1 submitted to Genbank(Accesion Number: S77867). In these primers, GTCGAC and GCTAGC indicatethe SalI and NheI site respectively. Ex Taq (Takara) was admixed with anequal amount of Taq Start Antibody (Clontech Laboratories, Inc.) toprevent amplification of nonspecific products and primer dimers.Reaction mixture was prepared by adding 5 μl of the buffer attached toEx Taq, 4 μl of dNTPs, 1 μl of the mixed solution of Ex Taq and TaqStart Antibody, and 1 μl of 50 μM each primers. The cDNA was diluted toone fifth with distilled water, and an aliguot (5 μl) was added to thereaction mixture. PCR conditions were as follows: denatured at 95° C.for 2 minutes, followed by 27 cycles at 95° C. for 30 seconds, 65° C.for 30 seconds and 72° C. for 1 minutes, and after these cycles at 72°C. for 7 minutes.

The PCR products were separated with 1.2% agarose gel and stained withethidium bromide. Slices of agarose gel containing the band about 1.1kbp were cut out with razor blade, and then filtered using an Ultra Freefilter unit (Millipore). The eluent was extracted with phenol:chloroform and precipitated in ethanol. The amplified DNA was subclonedinto pCR™II with a TA cloning Kit (Invitrogen Co.), and then introducedinto E. coli JM109 competent cells. Transformants were selected in LB(Luria-Bertani) agar culture medium containing ampicillin, IPTG(isopropylthio-beta-D-galactoside), and X-gal(5-bromo-4-chloro-3-indolyl-beta-D-galactoside). The individual cloneswere cultured in an LB culture medium containing ampicillin and treatedwith an automatic plasmid extracting machine (Kurabo) to prepare plasmidDNAs respectively. Sequencing was carried out with a ABI PRISM DyeTerminator Cycle Sequencing Kit FS (Perkin-Elmer), and an ABI automaticsequencer. In the FIG. 52, underlines indicate the sequencescorresponding to the parts of primer sequences. Double-lined basesindicate the base substitution compared with the sequence data reported,and one of these substitutions was accompanied by an amino acidsubstitution from ²⁸⁹Leu(CTC) to ²⁸⁹Val(GTC). A plasmid, pCRII-UHR-1,containing the UHR-1 cDNA fragment was thus constructed.

UHR-1 cDNA expression plasmid was prepared as follows. First,pCRII-UHR-1 was digested with NheI and SalI. The resultant fragment ofabout 1.1 kbp was separated through electrophoresis using a 1.2% agarosegel and precipitated as above. The DNA fragment was then ligated intothe NheI-SalI site of pAKKO-111H, with a Ligation System (Takara). Aresultant expression plasmid, pAKKO-UHR-1 was introduced into E. coliJM109.

CHO dhfr⁻ cells were grown in 10 cm diameter Petri dishes at the cellnumber of 1×10⁶, and cultured at 37° C. for 24 hours in α-MEM containing10% of fetal bovine serum. The expression plasmid (20 μg) was introducedinto the cells by a liposome method using a Gene Transfer (Nippon Gene).After 24 hours from the introduction, the medium was substituted withfresh one. After additional 24 hour incubation, the culture medium waschanged to a Selection medium, α-MEM without nucleosides containing 10%of dialyzed fetal bovine serum. Culture was carried out until cellsgrowing in the Selection medium were obtained. CHO-UHR-1 which highlyexpressed UHR-1 was thus established.

Example 34

Radioiodination of 19P2-L31 and Receptor Binding Experiments

19P2-L31 was radioiodinated with [¹²⁵I]-Bolton-Hunter Reagent(NEN.Dupont; NEX-120) as follows. Two hundred microliter of[¹²⁵I]-Bolton-Hunter Reagent was dried in a 500 μl Eppendorf tube withN₂ gas. The dried reagent was dissolved in 2 μl of acetonitrile, andthen mixed with 4 ml of 50 mM phosphate buffer (pH 8.0) and 4 μl of19P2-L31 3×10⁻⁴M The mixture was incubated at room temperature for 40min and the reaction was stopped by adding 5 μl of 1.0 M glycine. Theall reaction mixture was diluted with 300 μl of 18% acetonitrile andinjected onto reverse-phase HPLC column TSK gel ODS-80™ (4.6×100 mm;TOSO). The radioiodinated 19P2-L31 was eluted with a linear gradient ofacetonitrile concentration from 18 to 32.4% in 0.1% teifluoroacetic acidfor 24 min at a flow rate of 1 ml/min. The peak fraction ofradioiodinated 19P2-L31 was collected and diluted with twice volume of50 mM Tris-HCl (pH7.5) containing 0.1% BSA and 0.05% CHAPS, and thenstored at −20° C.

Receptor binding experiments were performed with [¹²⁵ I]-19P2-L31 asfollows. As receptor-expressing CHO cells, CHO-19P2-9; mono-clone ofCHO-19P2, CHO-UHR-1, and mock CHO were used in this experiment.CHO-19P2-9 cells are ones selected from CHO-19P2 cells by ultradilutiontechnique using 96-well microplate as clone which indicated strongerarachidonic acid metabolic-release promoting reaction by 19P2-L31. Themock CHO cells are ones for control which were transformed withexpression vector pAKKO alone. These cells cultured in flasks forculturing tissues were harvested with 5 mM EDTA/PBS, and thenresuspended in HBSS containing 0.05% BSA and 0.05% CHAPS at 0.5×10⁷cells/ml. The cell suspensions were incubated with 200 pM[¹²⁵I]-19P2-L31 for 2.5 hr at room temperature in a 100 μl total volume.The reaction mixture were diluted with 2 ml of an ice-cold beffer (50 mMTris-HCl pH7.5 containing 5 mM EDTA, 0.05% BSA, and 0.05% CHAPS) andimmediately filtered though glass filters GF/F (Whattman) which werepre-wetted with the buffer containing 0.3% polyethylenimine. The glassfilters were subjected to γ-counting. Non-specific binding wasdetermined in the presence of 200 nM unlabeled 19P2-L31.

[FIG. 36] shows receptor binding experiments with [¹²⁵I]-19P2-L31 onlive cells.

Specific binding of [¹²⁵I]-19P2-L31 was detected on CHO cells which wereexpressed with hGR3 and rat homolog UHR-1 respectively. The experimentswere performed in triplicate. These results show that the proteinsencoded by hGR3 and UHR-1 is functioning as the specific receptor of19P2-L31.

Example 35

Release of Arachidonic Acid Metabolites from CHO-19P2-9 and CHO-UHR1 by19P2-L31

Same as described in Example 11, the release activity of arachidonicacid metabolite was measured on CHO-19P2-9 and CHO-UHR1 and mock CHO.

[FIG. 37] shows the release activity of arachidonic acid metabolite onCHO-19P2-9 and CHO-UHR1 by 19P2-L31.

On CHO cells which were expressed with rat homolog UHR1, the releaseactivity of arachidonic acid metabolite was detected same as CHO-19P2-9.The experiments were performed in duplicate. These results show that theprotein encoded by UHR-1 is functioning as the specific receptor as wellas hGR3.

Example 36

Quantification of Rat 19P2 Ligand and Rat UHR-1 mRNA, BBRC, 209,606-613,1995) by RT-PCR

(1) Preparation of Poly(A)+RNA and cDNA Synthesis from Rat tissues.

Poly(A)+RNA was isolated from a variety of tissues in rats (Wisterstrain, male, 8 weeks old) by homogenization with Isogen (Nippon Gene)followed by an oligo (dT)-cellulose chromatography (Pharmacia). One μgof poly(A)+RNA was treated with DNase I (Amplification grade, GibcoBRL)to eliminate the contamination of genomic DNA. DNase I was inactivatedby the addition of 25 mM EDTA solution at 65° C. Then RNA (160 ng) wasreverse-transcribed in 4011 of a reaction miexture containing 10 mM ofTris-HCl (pH 8.3), 2.5 μM of random hexamers (Takara), 0.4 mM of eachdNTP, and 10 U of AMV reverse transcriptase XL (Takara). The sampleswere incubated at 30° C. for 10 min followed by 42° C. for 1 h, then 99°C. for 5 min to stop the reaction. The reaction mixture was purified byethanol precipitation, and then the cDNA was diluted to 40 μl withtricine-EDTA buffer (correspond to 4 ng poly(A)+RNA/μl).

(2) Construction of Positive Control Plasmid Vectors

Rat glycerolardehyde-3-phasphate-dehydrogenase (G3PDH) and rat UHR-1cDNAs were isolated from rat pituitary tumor cell line GH₃ by means ofRT-PCR Poly(A)+RNA of GH₃ was prepared by FastTrack (Invitrogen), andcDNA was synthesized as Example 36(1). Oligonucleotide primers used forthe amplification are as follows: rat G3PDH amplification primer set(Clontech), rRECF (5′-CCTGCTGGCCATTCTCCTGTCTTAC-3′) (SEQ ID NO:88) andrRECR (0.5′-GGGTCCAGGTCCCGCAGAAGGTTGA-3′) (SEQ ID NO:89) for UHR-1. Thefragments amplified from GH₃ cDNA were subcloned with a TA cloning Kit(Invitrogen). The recombinant vectors were introduced into E. coliJM109. The transformant clones were cultured in a LB culture mediumcontaining ampicillin, and the plasmid DNAs were prepared with a QuiagenPlasmid Midi Kit (Quiagen). The plasmid of rat ligand polypeptide wasprepared from E. coli JM109/pRAV3 which was deposited.

(3) Quantification RT-PCR

cDNA and plasmid DNA prepared in (1) and (2) above were diluted withdistilled water to adequate concentrations and used as templates ofquantitative RT-PCR. G3PDH, UHR-1, and ligand polypeptide cDNA fragmentswere amplified using human G3PDH amplimer (Clontech), rRECF and rRECR,and r19F (5′-GAAGACGGAGCATGGCCCTGAAGAC-3′) (SEQ ID NO:91) and r19R(5′-GGCAGCTGAGTTGGCCAAGTCCAGT-3′) (SEQ ID NO:91), respectively. Eachreaction sample contained 100 μM of dNTP mixture, 200 nM of each primer,4 μl of template DNA, 0.25 μl of 50× KlenTaq DNA polymerase mix(Clontech), and 2.5 μl of the buffer attached to KenTaq DNA polymerasemix in a final volume of 25 μl. PCR conditions for G3PDH were asfollows: denatured at 94° C. for 1 min, followed by 26 cycles at 98° C.for 10 sec, at 65° C. for 20 sec, and at 72° C. for 40 sec. PCRconditions for UHR-1 and ligand polypeptide were as follows: denaturedat 94° C. for 1 min, followed by 34 cycles at 98° C. for 10 sec, and at68° C. for 25 sec. An aliquot 5 μl of each RT-PCR product was separatedwith 4% Nusieve 3:1 agarose gel (F.M.C.) electrophoresis and stainedwith ethidium bromide. The bands were quantified using a densitometryprogram (Advanced American Biotechnology).

The results measured the expression levels of UHR-1 and ligandpolypeptide mRNA in the tissues were shown in FIGS. 38 and 39respectively. UHR-1 and ligand polypeptide mRNA were detected in all thetissues tested. The highest level of UHR-1 mRNA expression was detectedin the pituitary, and moderate expression levels in the brain, whereaspoorly expressed in peripheral tissues except for the adrenal glands.Ligand polypeptide mRNA expressed mainly in the hypothalamus and dosalmedulla among brain regions, and expressed comparatively high levels inthe lung, thymus, pancreas, kidney, adrenal glands, and testis. Theseresults show that the UHR-1 and ligand polypeptide play a significantrole for the regulation of function in various tissues.

Example 37

Effect of 19P2-L31 on Glucose-Induced Increase in Plasma InsulinConcentration

Male Wistar rats (8-10 w) were anesthetized by i.p. injection ofpentobarbital (65 mg/kg). Glucose alone (86 mg/rat) or glucose and19P2-L31 (675 pmol, 2.25 nmol, 6.75 nmol and 67.5 nmol/rat) wereadministered by bolus injection in the jugular vein. Blood samples werewithdrawn from the contralateral vein. Plasma insulin concentration wasdetermined with a radioimnunoassay kit (Amersham).

Administration of 19P2-L31 at the doses of 675 pmol, 2.25 nmol, and 6.75nmol partially inhibited glucose-induced sharp increase (the firstphase) in plasma insulin concentration at 2 min postinjection and theblunt increase (the second phase) after 6 min postinjection. Itcompletely inhibited the first and second phase of increase in insulinconcentration at the dose of 67.5 nmol [FIG. 40].

Example 38

Effects of Ligand Polypeptide on Motor Activity of Mouse

The effects of administration of 19P1-L31 to mouse lateral ventricle onmotor activity were studied. The mature ICR male mice (weight atoperation: about 35 g) were anesthetized by intraperitonealadministration of 50 mg/kg of pentobarbital, and then fixed on astereotaxic apparatus. The skull of a said mouse was exposed, then ahole was made by dental drill for guide-cannulization into the leftlateral ventricle. The tip of a stainless-steel guide-cannula (24G,length: 5 mm) for drug injection to lateral ventricle, was inserted tothe position of AP: +0.6 mm (from bregma), L: left 1 mm and H: −1 mm(from dura matter). The guide-cannula was fixed onto the skull withadhesive. The cannula-implanted mice were housed as described above andwere used for behavioral analysis at least 3 days after the operation.

Motor activity such as spontaneous motor activity and rearing wasmeasured while each mouse was in a transparent acrylic cage (24×37×30cm) within a soundproofed, illuminated (light up: at 6-18 o'clock) box.Tap water and laboratory chow were available ad libitum. Motor activitywas measured by means of a Supermex (Muromachi Kikai). Drugs and PBSwere administered at 2:30±30 p.m. At the administration, astainless-steel micro-injection cannula (30G, length: 6 mm) was insertedinto the guide-cannula. The micro-injection cannula was connected to amicrosyringe pump with Teflon tube, and injection of PBS or a peptidedissolved in PBS lasted for 2 minutes at a speed of 2 μl/min. Themicro-injection cannula was withdrawn after over a period of 2 minutesfrom end of injection, then motor activity was meausred.

The results are expressed as a mean±S.E.M. Student's t test was used todetermine the significance of differences between values from the micetreated with a peptide and the PBS-injected controls. For the purpose ofthis analysis, p<0.05 was assumed to be the minimal level ofsignificance.

As shown in [FIG. 41], administration of 10 nmol of 19P2-L31 caused asignificant increase in spontaneous motor activity at 70-105 min afterinjection. Rearing behavior also showed significant variation. While theadministration of 1 nmol of 19P2-L31 did not cause statisticallysignificant change of spontaneous motor activity, rearing behaviorshowed a significant decrease at only 105 min after injection [FIG. 42].The administration of 0.1 nmol of 19P2-L31 caused a significant increaseat 25 min, 40 min and 70 min after injection. In that case, rearingbehavior showed an increasing tendency similarly to spontaneous motoractivity, however that was not statistically significant [FIG. 43]. Theadministration of 0.01 nmol of 19P2-L31 caused a significant increase at20 min and 40 min after injection. In that case, rearing behavior showedan increasing tendency similarly to spontaneous motor activity, howeverthat was not statistically significant [FIG. 44].

Example 39

Effects of Ligand Polypeptide on Reserpine-Induced Hypothermia in Mice

The mature ICR male mice (weight at operation: about 35 g) wereanesthetized by administration of pentobarbital (50 mg/kg, i.p.), andthen fixed on stereotaxic apparatus. The skull of a said mouse wasexposed, then a hole was made by dental drill for guide-cannulizationinto the left lateral ventricle. The tip of a stainless-steelguide-cannula (24G, length: 5 mm) for drug injection to lateralventricle, was inserted to the position of AP: +0.6 mm (from bregma), L:left 1 mm and H: −1 mm (from dura matter). The guide-cannula was fixedonto the skull with adhesive. The cannula-implanted mice were housed asdescribed above and were used for measurements of body temperature atleast 3 days after the operation. Reserpine (Apoplon; DaiichiPharmaceutical) was administered to mice at a dose of 3 mg/kg, s.c., andafter 15 hours, each mouse was placed in a cage for the measurement.Then a stainless-steel micro-injection cannula (30G, length: 6 mm) wasinserted into the guide-cannula. The micro-injection cannula wasconnected to a microsyringe pump with Teflon tube, and injection of PBSor a peptide dissolved in PBS lasted for 2 minutes at a speed of 1μl/min. The micro-injection cannula was withdrawn after over a period of2 minutes from end of injection, then the temperature in rectum wasmeasured.

The results are expressed as a mean±S.E.M. Student's t test was used todetermine the significance of differences between values from the micetreated with a peptide and the PBS-injected controls. For the purpose ofthis analysis, p<0.05 was assumed to be the minimal level ofsignificance.

As shown in (FIG. 45), body temperature which was lowered by reserpineincreased significantly after a 10 nmol injection of 19P2-L31 incontrast to the control which PBS were administered. This increase ofbody temperature reached a maximum level at 45 min after administrationof the peptide. On the other hand, there was no statisticallysignificant difference in temperature variation between 1 nmol of19P2-L31 and the PBS-injected control throughout the experimentalperiod.

Example 40

Effects of Ligand Polypeptide on Blood Pressure in Rats

The inventors explored the influence of injection of 19P2-L31 into thearea postrema of medula oblongata on blood pressure. Mature male Wistarrats (body weights at operation: ca 300 g) were anesthetized withpentobarbital 50 mg/kg i.p. and each animal was immobilized in a ratbrain stereotaxic apparatus. The incisor bar was lowered by 3.3 mm fromthe interaural line. The skull was exposed, and using a dental drill ahole was made on the skull for implantation of a guide cannula. Inaddition, anchor screws were buried in two positions around the drilledhole. A stainless-steel guide cannula, AG-12 (0.4 mm inside dia., 0.5 mmout. dia., EICOM), was inserted in such a manner that its leading endwould be situated in the upper part of the area postrema. For thispurpose, the guide cannula was instered from a forward direction at anangle of 200 with the perpendicular (FIG. 46; Note, however, that thedrawing shows a microinjection cannula 1.0 mm longer than the guidecannula). With reference to the atlas of Paxinos and Watson (1986), thestereotaxic coordinates were AP: −6.0 mm (from interaural line), L: 0.0mm, and H: +1.5 mm (from interaural line). The guide cannula was securedto the skull using an instant adhesive, a dental cement, and anchorpieces. A stainless-steel dummy cannula, AD-12 (0.35 mm out. dia.,EICOM), was inserted into the guide cannula and locked in position witha cap nut (EICOM). Thereafter, the rats were kept in individual cages.

About a week of feeding after implantation of the guide cannula forpostoperative recuperation, an operation was performed for measurementsof blood pressure in conscious state. The rat described above wasanesthetized with pentobarbital 50 mg/kg i.p. and immobilized in spineposition on a necropsy pad and the left femoral artery was exposed.Polyethylene tubing, SP35 (0.5 mm in. dia., 0.9 mm out. dia., NatsumeSeisakusho), was cut to about 60 cm in length and filled with 200 U/mlheparin-containing saline. This tube was inserted about 2.5 cm deep intothe femoral artery and secured in position. The free end of the tube waspassed under the dorsal skin and exposed in the cervical region (dorsalside).

After waiting overnight postoperatively, the polyethylene tube wasconnected to a transducer (Spectramed) and the blood pressure wasmeasured. After blood pressure readings became steady, the cap nut anddummy cannula were removed from the rat skull and, instead, a stainlesssteel microinjection cannula (0.17 mm in. dia., 0.35 mm out. dia.,EICOM) connected to a Teflon tube (50 cm long, 0.1 mm in. dia., 0.4 mmout. dia., EICOM) was inserted. The length of the microinjection cannulawas adjusted beforehand so that its tip would extend 1 mm from the guidecannula (FIG. 46). One end of the Teflon tube was connected to amicrosyringe pump and either PBS or 19P2-L31 dissolved in PBS wasinjected, in a total volume of 2 μl, into the area postrema at a flowrate of 1.0 μl/min.

After measurement of blood pressure, the micro-injection cannula usedfor injection of 19P2-L31 was disconnected and replaced with amicroinjection cannula for injection of a stain (Evans Blue) solution.The stain was infused at the same rate of 1.0 μl/min as the injection of19P2-L31 for 2 minutes. After a standby time of about 3 minutes, themicroinjection cannula was disconnected. The rat was decapitated and thebrain was quickly removed and frozen. The brains were cut serial frontalsections on cryostat and the position of dye infusion was confirmed.

Results of the above experiment showed that injection of 10 nmol of19P2-L31 into the area postrema of medula oblongata caused an elevationof blood pressure. Typical examples of direct and mean blood pressureare shown in FIG. 47.

Example 41

Effects of Ligand Polypeptide on Plasma Pituitary Hormone Level

The inventors explored the effect of 19P2-L31 administered into thethird ventricle on pituitary hormone levels in the plasma. Mature maleWistar rats (body weights at operation: 290-350 g) were anesthetizedwith pentobarbital, 50 mg/kg i.p., and each immobilized in a rat brainstereotaxic apparatus. The incisor bar was set 3.3 mm lower from theinteraural line. The skull was exposed, and using a dental drill a holewas made on the bone for implantation of a guide cannula. In addition,an anchor screw was buried in one position around the hole. Astainless-steel guide cannula, AG-12 (0.4 mm in. dia., 0.5 mm out. dia.,EICOM), was inserted in such a manner that its tip would be situated inthe upper part of the third ventricle. With reference to the atlas ofPaxinos and Watson (1986), the stereotaxic coordinates were AP: +7.2 mm(from interaural line), L: 0.0 mm, and H: +2.0 mm (from interauralline). The guide cannula was secured to the skull using an instantadhesive, a dental cement, and an anchor piece. A stainless-steel dummycannula, AD-12 (0.35 mm out. dia., EICOM), was then passed through theguide cannula and locked in position with a cap nut (EICOM). After theoperation the rats were housed in individual cages and kept for at least3 days for recuperation before starting the experiment.

The operated rat was anesthetized with pentobarbital 50 mg/kg i.p. andimmobilized in dorsal position. After the bilateral jugular veins wereexposed, 400 μl of blood was drawn using a 1 ml tuberculin syringe and a24-G needle (both by Termo). To prevent clotting, the syringe was filledwith 20 μl of saline containing 200 U/ml of heparin beforehand. The capnut and dummy cannula were removed from the rat skull and, instead, astainless steel microinjection cannula (0.17 mm in. dia., 0.35 mm out.dia., EICOM) connected to Teflon tube (50 cm long, 0.1 mm in. dia., 0.4mm out. dia., EICOM) was inserted. The length of the microinjectioncannula was adjusted beforehand so that its tip would be emergent fromthe guide cannula by 1 mm. One end of the Teflon tube was connected to amicrosyringe pump and either PBS or 19P2-L31 dissolved in PBS wasinjected, in a total volume of 10 μl, into the third ventricle at a flowrate of 2.5 μl/min. After a standby time of 1 minute following infusion,the microinjection cannula was disconnected and the dummy cannula wasreinstated and locked in position with a cap nut. Immediately beforeinitiation of intraventricular administration and 10, 20, 30, 40, and 60minutes after initiation of administration, 400 μl portions of bloodwere drawn from the jugular vein. Each blood sample was centrifuged(5,000 rpm, 10 min.) with a high-speed refrigerated microcentrifuge(MR-150, Tommy Seiko) and the supernatant (plasma) was recovered. Theamounts of pituitary hormones [prolactin, luteinizing hormone (LH),adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH),and growth hormone (GH)] in the plasma were respectively determined byradioimmunoassays.

The results were expressed as a mean±S.E.M. To test for significantdifference between the group treated with 19P2-L31 dissolved in PBS andthe control group treated with PBS alone, Student's t-test was used.According to the two-tailed test, p<0.05 was assumed to be the minimallevel of significance. As shown in FIG. 48, the plasma GH level wassignificantly decreased at 20 minutes after administration of 50 nmol of19P2-L31 into the third ventricle, as compared with the control group.Tendencies toward decrease were found at 10, 30, and 40 minutes afteradministration as well but the changes were not statisticallysignificant. At 60 minutes after administration, there was no differencefrom the control group. As to plasma prolactin, LH, ACTH, and TSH, noneshowed significant changes.

Example 42

Effects of Ligand Polypeptide on plasma growth Hormone (GH) Level inFreely Moving Rats

Mature male Wistar rats were anesthetized with pentobarbital 50 mg/kgi.p. and, as in Example 41, a stainless-steel guide cannula AG-12 (0.4mm in. dia., 0.5 mm out. dia., EICOM) was implanted in position with itstip situated in the upper part of the third ventricle. After theoperation the rats were housed in individual cages and kept for at least3 days for recuperation and, then, a cannula (30 cm long, 0.5 mm in.dia., 0.9 mm out. dia., Natsume Seisakusho) filled with heparin (200U/ml)-containing saline was inserted into the right atrium from theright jugular vein under pentobarbital anesthesia. The rats weremaintained-overnight for complete arousal from anesthesia and thentransferred to transparent acrylic cages (30 cm×30 cm×35 cm). A 1 mltuberculin syringe with a 24-G needle (both by Termo) was connected tothe cannula inserted in the atrium and 300 μl of blood was drawn. Toprevent clotting, the syringe was filled with 20 μl of saline containing200 U/ml of heparin beforehand. A stainless-steel microinjection cannula(0.17 mm in. dia., 0.35 mm out. dia., EICOM) connected to Teflon tube(50 cm long, 0.1 mm in. dia., 0.4 mm out. dia., EICOM) was inserted intothe guide cannula positioned in the third ventricle. The length of themicroinjection cannula was adjusted beforehand so that its tip would beextend 1 mm from the guide cannula. One end of the Teflon tube wasconnected to a microsyringe pump and either PBS or 19P2-L31 dissolved inPBS was injected, in a total volume of 10 μl, into the third ventricleat a flow rate of 2.5 μl/min. Ten minutes after initiation ofadministration into the third ventricle, 5 μg/kg GHRH-saline wasadministered via the cannula inserted into the atrium. Immediatelybefore initiation of intraventricular administration and 10, 20, 30, 40,and 60 minutes after administration of GHRH, 300 μl portions of bloodwere drawn from the jugular vein. Each blood sample was centrifuged(5,000 rpm, 10 min.) and the supernatant (plasma) was recovered. Theconcentrations of GH in the plasma were determined by radioimmunoassay.

The results were expressed as a mean±S.E.M. To test for significantdifference between the group treated with 19P2-L31 dissolved in PBS andthe control group treated with PBS alone, Student's t-test was used.According to the two tailed test, p<0.05 was assumed to be the minimallevel of significance. As shown in FIG. 49, administration of 5 μg/kg ofGHRH elevated the plasma GH level. However, when 50 nmol of 19P2-L31 wasadministered into the third ventricle, the GHRH-induced elevation ofplasma GH was significantly inhibited.

Example 43

Preparation of Rabbit Anti-Bovine 19P2-L31 Antibodies

Synthetic peptides containing partial 19P2-L31 sequence (peptide-I:SRAHQHSMEIRTPDC (SEQ ID NO:92), peptide-II: CAWYAGRGIRPVGRFNH₂ (SEQ IDNO:93), and peptide-III: CEIRTPDINPAWYAG (SEQ ID NO:94) were conjugatedwith KLH according to the standard method. Each peptide conjugate (600μg as a peptide) dissolved in saline was mixed with Freund's completeadjuvant, and the resultant emulsion was subcutaneously injected intothree rabbits (NZW, male, 2.5 kg) respectively. Hyperimmunization wascarried out three times in total at the same dose of the conjugate asthe first injection with Freund's imcomplete adjuvant every three weeks.Antibody titers were determined as follows. Two weeks after the lastimmunization, blood samples were obtained from the vein of the immunizedrabbits respectively. After being incubated at 37° C. for 1 hour, theblood samples were kept at 4° C. over night. Sera were then prepared bymeans of centrifugation. An aliquot (100 μl) of each serum samplediluted properly was introduced into 96-well polystyrene microplateswhich were pre-coated with goat anti-rabbit IgG (Fc) antibodies, andthen the microplates were incubated at 4° C. for 16 hours. Afterremoving the sera, horse radish peroxidase (HRP)-conjugated peptide-I,II, and III were added to the wells respectively, and then themicroplates were incubated at room temperature for 4 hours. Afterremoving the peptides, coloring reaction was done by adding a substrate.The reaction was stopped by adding 100 μl of a stopping solution, andthen the absorbance at 450 nm in each well was measured. As shown inFIG. 50, serum samples obtained from the rabbits after the immunizationshowed binding activities to HRP-conjugated peptides respectively.However, none of binding activities was detected in sera prepared beforethe immunization. These results indicated that the rabbits received theimmunization produced antibodies against peptide-I, II, and III,respectively. To prepare purified IgG antibody fractions, sera obtainedfrom the immunized rabbits was percipitated with anmonium sulfate. Theresultant precipitates were dissolved in borate buffer, and thendialyzed with the same buffer. The IgG fractions thus obtained were thensubjected onto affinity columns conjugated with peptide-1 or 19P2-L31respectively. After washing the columns with borate buffer and followingwith acetate buffer (100 mM, pH 4.5), antibodies bound to the columnwere eluted with glycine buffer (200 mM, pH 2.0). After beingneutralized with 1M Tris, the eluents were used as purified antibodiesrespectively.

Example 44

Inhibitory Activity of Antibodies Against the Release of ArachidonicAcid Metabolites Induced by 19P2-L31

The purified antibodies prepared as described in Example 43 were testedtheir inhibitory activity against the release of arachidonic acidmetabolites induced by 19P2-L31. The antibodies diluted as indicated inFIG. 51 were mixed with 19P2-L31 (5×10⁻¹⁰M) at room temperature for 1hour, and then the release of arachidonic acid metabolites was examinedas described in Example 11. As shown in FIG. 51, the highest inhibitoryactivity was observed in anti-peptide-II antibodies.

Preparation Example 1

Fifty milligrams of the compound as obtained in Example 21 is dissolvedin 50 ml of Japanese pharmacopoeial, distilled water for injection, andJapanese pharmacopoeial, distilled water for injection is added theretoto make 100 ml. The resulting solution is filtered under a germ-freecondition, and the filtrate of 1 ml each is filled in vials forinjection, freeze-dried and sealed therein also under a germ-freecondition.

Preparation Example 2

One hundred milligrams of the compound as obtained in Example 21 isdissolved in 50 ml of Japanese pharmacopoeial, distilled water forinjection, and Japanese pharmacopoeial, distilled water for injection isadded thereto to make 100 ml. The resulting solution is filtered under agerm-free condition, and the filtrate of 1 ml each is filled in vialsfor injection, freeze-dried and sealed therein also under a germ-freecondition.

[Evaluation of the Physiological Activities of Ligand Polypeptide of thePresent Invention]

The above examples 37-41 demonstrate that topical administration ofligand polypeptide induces enhancement of spontaneous motor activity andrearing behavior, elevation of body temperature and blood pressure, anddecrease in plasma growth hormone concentration. These findings relatingto physiological activities are the first proof of various prominentphysiologic changes which occur when ligand polypeptide acts on thecentral nervous system.

Since ligand polypeptide of the present invention, inclusive of itssalt, acts on the central nervous systems of warm-blooded animals (e.g.rat, mouse, guinea pig, chicken, rabbit, dog, swine, bovine, sheep,monkey, and man) to induce a variety of pharmacological changes, it isshowed that the ligand and salt have the property to alter theintracranial nervous system and endocrine system.

When 19P2-L31 was administered into the lateral ventricle of mice, anincrease in the amount of activity was found at the level of 0.01-10nmol. This fact shows that ligand polypeptide triggers changes in themotor system via the G protein-coupled receptors of the central nervoussystem. It was also found that administration of the peptide into thelateral ventricle of mice results in elevation of body temperature andthat administration into the area postrema of medula oblongata of ratsresults in elevation of blood pressure. These actions resemble thepharmacologic actions of known central stimulants (e.g. amphetamine,cocaine, methylphenidate, etc.). Therefore, it is showed that ligandpolypeptide or a salt thereof releases biologic amines (dopamine,noradrenaline, serotonin) from the nerve ending reservoirs, in the main(Michio Yuzuru and Takeo Yoshikawa, Medical Science, 42, 535-536, 1991).

Furthermore, when 19P2-L31 was injected into the third ventricle ofrats, the plasma growth hormone level was depressed. This finding showsthat this peptide acts on the hypothalamus and is associated withsecretion of pituitary hormones via the hypothalamopituitary system. Itis also possible that this peptide directly act on the pituitary so asto suppress the release of growth hormone. Growth hormone releasinghormone (GHRH) which regulates secretion of growth hormone from thehypophysis as well as somatostatin exists in the neighborhood of thethird ventricle (Masahiro Tohyama et al., Kagakuteki ShinkeikinoKaibogaku (Chemical Neuroanatomy), 167-216, 1987). Therefore, it isshowed that 19P2-L31 is modulating release of these substances.

The above facts show that ligand polypeptide is a peptide acting on thecentral nervous system to control the autonomous nervous system. Thefact that the mRNA of this peptide and of its receptor is expressed athigh levels in the hypothalamus and medula oblongata also shows theinvolvement of ligand polypeptide in the modulation of the autonomousnervous system. In fact, the superior center of autonomous nerveperipherals is the medula oblongata and hypothalamus, where as alreadyelucidated the sympathetic nervous system and the parasympatheticnervous system are integrated to play an important role in both neuralregulation and humoral regulation.

The above findings indicate the usefulness of ligand polypeptide or anagonist of ligand polypeptide, or a salt thereof, as a central nervoussystem stimulant causing enhancement of spontaneous motor activity.Thus, the peptide can be used as a prophylactic and/or therapeutic drugfor a variety of diseases such as senile dementia, cerebrovasculardementia (dementia due to cerebrovascular disorder), dementia associatedwith phylodegenerative retroplastic diseases (e.g. Alzheimer's disease,Parkinson's disease, Pick's disease, Huntington's disease, etc.),dementia due to infectious diseases (e.g. delayed viral infections suchas Creutzfelt-Jakob disease), dementia associated with endocrine,metabolic, and toxic diseases (e.g. hypothyroidism, vitamin B12deficiency, alcoholism, and poisoning due to various drugs, metals, ororganic compounds), dementia associated with oncogenous diseases (e.g.brain tumor), dementia due to traumatic diseases (e.g. chronic subduralhematoma), depression (melancholia), hyperkinetic (microencephalopathy)syndrome, or disturbance of consciousness. On the other hand, anantagonist of 19P2 ligand or a salt thereof is of value as a CNSdeppressant, for instance, and can be used as an antipsychotic drug, ananti-Huntigton's disease drug, an antianxiety drug, or ahypnotic-sedative.

It was made clear that injection of ligand polypeptide into the areapostrema of medula oblongata elevates the blood pressure. Therefore,ligand polypeptide or an agonist of ligand polypeptide, or a saltthereof, is of value as a vasopressor. On the other hand, a ligandpolypeptide antagonist or a salt thereof is of value as a depressor.

It was found that when ligand polypeptide acts on the hypothalamus, theplasma growth hormone level is depressed. Hypersecretion of growthhormone triggers somatomegaly and acromegalic gigantism (Katamasu etal., Endocrine Syndrome, 78-80, 1993; Hiroi et al., Endocrine Syndrome,149-151, 1993). Therefore, ligand polypeptide or a ligand polypeptideantagonist, or a salt thereof, can be used as a prophylactic and/ortherapeutic drug for somatomegaly and acromegalic gigantism. Moreover,growth hormone promotes release of glucose from the liver and inhibitsthe uptake of glucose by muscles and adipose tissues from the blood,causing hyperglycemia and diabetes [Eiji Kobayashi, Naibumpi Gensho(Endocrine Phenomena), 1980]. In fact, the secretion of growth hormoneis elevated in diabetic patients (Hiroshi Kiyono, Endocrinology andMetabolic Diseases, 385-402, 1994). Therefore, ligand polypeptide or anagonist of ligand polypeptide, or a salt thereof, can be used as aprophylactic and/or therapeutic drug for diabetes, for instance.

On the other hand, an antagonist of ligand polypeptide promotessecretion of growth hormone. Therefore, a ligand polypeptide antagonistor a salt thereof can be used as a prophylactic and/or therapeutic drugfor pituitarism leading to a depressed growth hormone level, pituitarydwarfism, and hypoglycemia. Moreover, growth hormone and insulin-likegrowth factor secreted by growth hormone are effective in amyotrophiclateral sclerosis, osteoporosis, renal failure, and improvement inpostoperative nutritional status (Shizume et al., Endocrine Syndrome,84-87, 1993, Nikkei Bio-Annal 96, 453-454, 1996; Tobiume et al.,Clinical Endocrinology, 44, 1205-1214, 1996). Therefore, a ligandpolypeptide antagonist or its salt can be used as a prophylactic and/ortherapeutic drug for such illnesses.

1-16. (canceled)
 17. A screening method for a compound capable ofchanging the binding activity of a ligand polypeptide comprising theamino acid sequence of SEQ ID NO:73, or its amide or ester, or a saltthereof, or a partial peptide thereof, with a receptor proteincomprising an amino acid sequence represented by SEQ ID NO: 22, or asalt thereof, or a partial peptide of said receptor protein, or a saltthereof, the method comprising making a comparison of said bindingactivity between: (i) at least one case where said polypeptide, or itsamide or ester, a partial peptide of said ligand polypeptide, or itsamide or ester, or a salt thereof is contacted with said receptorprotein or a salt thereof or a partial peptide of said receptor protein,or a salt thereof, and (ii) at least one case where said ligandpolypeptide or its amide or ester, or a salt thereof, or the partialpeptide of said ligand polypeptide, or its amide or ester, or a saltthereof, together with a sample containing the compound to be tested iscontacted with the receptor protein or a salt thereof or a partialpeptide of said receptor protein, or a salt thereof. 18-20. (canceled)21. The method as claimed in claim 17, wherein the ligand polypeptidecomprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO: 47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,SEQ ID NO:65, or SEQ ID NO:66.
 22. The method as claimed in claim 17,wherein the ligand polypeptide comprises the amino acid seqeunece of SEQID NO:1, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:59.